阅读说明：本技术 高表达TrkB作为新型靶点在抑制胰腺癌转移方面的医药用途 (Medical application of high-expression TrkB as novel target point in inhibiting pancreatic cancer metastasis ) 是由 张勇 魏炜 张昊 袁达伟 朱琨 李康 王直 郝楠 贾瑞 于 2019-10-15 设计创作，主要内容包括：本发明提出了高表达TrkB作为新型靶点在抑制胰腺癌转移方面的医药用途,例如TrkB的抑制剂(高表达TrkB)在制备用于抑制胰腺癌细胞转移的药物方面的用途等。本发明首次明确了：TrkB有促进胰腺癌细胞与骨髓迁徙干细胞抑或胰腺组织干细胞进行细胞融合形成肿瘤干细胞的能力,TrkB所介导的大聚集体外细胞互噬现象参与了转移的肿瘤细胞大聚集体结构完整性的维持,胰腺癌转移大聚集体可能通过释放高表达TrkB的微囊泡抑制机体的细胞免疫而达到免疫逃逸的目的；因此,通过抑制肿瘤转移大聚集体的形成或破坏已形成的大聚集体,可达到抑制胰腺癌转移的治疗效果。(The invention provides a medical application of high-expression TrkB as a novel target point in inhibiting pancreatic cancer metastasis, for example, an application of a TrkB inhibitor (high-expression TrkB) in preparing a medicament for inhibiting pancreatic cancer cell metastasis and the like. The invention first makes clear that: TrkB has the capacity of promoting pancreatic cancer cells and bone marrow migratory stem cells or pancreatic tissue stem cells to form tumor stem cells through cell fusion, the TrkB-mediated in-vitro cell interfusion phenomenon of large aggregates participates in maintaining the structural integrity of the large aggregates of the metastatic tumor cells, and the pancreatic cancer metastasis large aggregates can achieve the purpose of immune escape by releasing microvesicles with high expression of TrkB to inhibit the cellular immunity of organisms; therefore, by inhibiting the formation of large aggregates of tumor metastasis or destroying the already formed large aggregates, a therapeutic effect of inhibiting the metastasis of pancreatic cancer can be achieved.)

Use of an inhibitor of TrkB in the manufacture of a medicament for inhibiting pancreatic cancer cell metastasis, said inhibitor being capable of inhibiting high expression of TrkB.

2. The use of claim 1, wherein said inhibitor is selected from an RNA interference molecule or an antisense oligonucleotide directed against the TrkB coding sequence.

3. The use of claim 2, wherein the RNA interference molecule is a shRNA, siRNA, miRNA, or dsRNA.

4. The use according to claim 1, wherein the inhibitor acts on the ribosomal localization sequence of TrkB or its coding sequence.

5. A medicine for inhibiting pancreatic cancer cell metastasis contains a component capable of inhibiting high expression of TrkB.

6. A method of screening for an anti-pancreatic tumor therapeutic comprising:

(a) providing a tumor cell line or tumor culture expressing TrkB;

(b) contacting the candidate drug with the tumor cell line or tumor culture provided in step (a) as an administration group;

(c) detecting the expression level of TrkB in the administration group, and comparing the expression level of TrkB with the expression level of TrkB in a control group not administered with the candidate drug;

if the detection result shows that the expression level of TrkB in the administration group is obviously lower than that in the control group, the candidate drug can effectively inhibit pancreatic cancer cell metastasis.

7. Use of a biomarker or an identification unit thereof adapted to detect the level of TrkB expression in the construction of a medical device/reagent related to pancreatic cancer for early pancreatic cancer screening, early diagnosis, risk assessment, drug screening and/or efficacy assessment.

Technical Field

The application relates to a marker and a therapeutic target for pancreatic cancer metastasis.

Background

The existence of tumor stem cells with stem cell characteristics capable of self-renewal has been found and identified in almost all known tumors, and was first confirmed from the clinical practice point of view in 2010, but was still puzzled for its source, \ 32429.

Pancreatic cancer is a digestive system tumor with hidden, extremely dangerous and highly malignant diseases, has rapid development, early metastasis, low surgical resection rate, easy relapse and metastasis after surgery, is insensitive to conventional chemotherapy and radiotherapy, has extremely poor prognosis, and almost equal morbidity and mortality. For years, no breakthrough progress is made on the treatment, the 5-year survival rate is still less than 5%, and the overall survival rate is not remarkably improved in nearly 30 years, which has become a worldwide problem. In view of this, the basic biological elements and the evolution process of the pancreatic cancer malignant phenotype, the internal relation between the malignant proliferation phenotype and the metastasis potential thereof, need to be deeply explored, and the research on the unique molecular biological characteristics of pancreatic cancer, the analysis of the key elements influencing the metastasis thereof, and the design of a targeted blocking method for the inhibition thereof need to be started from the tumor invasion and metastasis multiple regulation mechanism, which becomes the important importance of the current research.

Trkb (tyrosine kinase receptor b) is a member of the neurotrophin receptor family (neuronal differentiation receptors), and it is specifically bound to the corresponding ligand BDNF (broad-derived neurotrophic factor) and has important significance in the aspects of biological evolution and development and cell survival, and it has been newly found that it can function independently of the corresponding ligand, and is an independent lethal factor and defined as a new protooncogene.

Disclosure of Invention

The applicant found that distant metastasis of pancreatic cancer depends on the formation of large aggregates of cancer cells mediated by TrkB, and tumor cells in the aggregates can counteract selective pressure from the environment and the occurrence of Anoikis by Autophagy (Autophagy). More interestingly, the tumor cells in the core appeared to have stem cell-like characteristics, which we found may be related to the induction of fusion of tumor cells highly expressing TrkB with stem cells derived from bone marrow migration or stem cells present in native tissues. And tumor cells around the aggregates appear to metastasize to the target organ by cooperating to become Trojan horses (Trojan horses) that protect the core stem cell-like tumor cells (e.g., it is possible to enhance aggregate integrity by mutually phagocytosing interphagy, release Microvesicles microviscles expressing TrkB to interfere with immunity, etc.). The research further verifies that the TrkB mediated cell fusion is a way for obtaining the characteristics of stem cells of tumor cells in pancreatic cancer metastasis aggregates on the basis of the previous research, and the inhibition on the TrkB signal pathway can inhibit the generation of tumor metastasis by interfering the cell fusion, destroying the structure of a large aggregate and releasing microvesicles.

Further, the applicant proposed for the first time:

(1) TrkB has the capacity of promoting pancreatic cancer cells and bone marrow migratory stem cells or pancreatic tissue stem cells to perform cell fusion to form tumor stem cells, and adds brand new evidence for the functional mechanism of the TrkB oncogene.

(2) The TrkB mediated in-vitro cell cross-feeding phenomenon of the large aggregates participates in the maintenance of the structural integrity of the large aggregates of the metastatic tumor cells, and provides an explanation for the survival of the tumor cells in the anoikis state.

(3) Pancreatic cancer metastasis large aggregates can achieve the aim of immune escape by releasing micro-vesicles highly expressing TrkB to inhibit cellular immunity of organisms.

(4) The formation of large aggregates is an essential morphological structure of tumor metastasis, and the inhibition of the large aggregates is a new way for resisting cancer.

(5) The method is important for researching the adhesion phenomenon between large aggregate cells and the mechanism of the large aggregate cells of tumor cells in the fluid dynamic state of anoikis and suspension.

Based on the above findings, the present application proposes the following medical fields:

in a first aspect, the use of an inhibitor of TrkB in the manufacture of a medicament for inhibiting pancreatic cancer cell metastasis, said inhibitor being capable of inhibiting high expression of TrkB (reducing the likelihood of high expression of TrkB and avoiding higher expression of already high-expressed TrkB). In particular, the anti-tumor therapeutic effect is achieved by inhibiting the formation of tumor metastasis large aggregates or destroying the formed large aggregates.

The inhibitor may be selected from an RNA interference molecule or an antisense oligonucleotide directed against the TrkB coding sequence.

Further, the RNA interference molecule is shRNA, siRNA, miRNA or dsRNA.

The inhibitor acts on the ribosome localization sequence of TrkB or its coding sequence.

In a second aspect, a medicament for inhibiting pancreatic cancer cell metastasis, which comprises a component capable of inhibiting high-expression TrkB.

In a third aspect, a method for screening an anti-pancreatic tumor therapeutic agent, comprising:

(a) providing a tumor cell line or tumor culture expressing TrkB;

(b) contacting the candidate drug with the tumor cell line or tumor culture provided in step (a) as an administration group;

(c) detecting the expression level of TrkB in the administration group, and comparing the expression level of TrkB with the expression level of TrkB in a control group not administered with the candidate drug;

if the detection result shows that the expression level of TrkB in the administration group is obviously lower than that in the control group, the candidate drug can effectively inhibit pancreatic cancer cell metastasis.

In a fourth aspect, the use of a biomarker or an identity module thereof suitable for detecting the level of TrkB expression in the construction of a medical device/reagent related to pancreatic cancer for early pancreatic cancer screening, early diagnosis, risk assessment, drug screening and/or efficacy assessment. The specific application method can refer to the method for screening the anti-pancreatic tumor therapeutic drugs, wherein the technical principle of detecting the TrkB expression level belongs to the conventional technical means.

Drawings

Fig. 1 and fig. 2 are schematic diagrams of experimental operation procedures for verifying that tumor cells inside a large aggregate formed when pancreatic cancer cells highly expressing TrkB are metastasized have stem cell characteristics.

FIG. 3 is a schematic diagram of the operation flow of the large aggregate structure stability-maintaining mechanism.

Fig. 4 is a schematic diagram of the operation procedure for verifying that TrkB microvesicles induce the release of myeloid-derived suppressor cells (MDSCs) and mediate T cell dysfunction.

FIG. 5 is a schematic diagram of the operation procedure of conventional research of TrkB mediated associated pathway and pancreatic cancer metastasis.

FIG. 6 is an electron microscope image of a metastatic tumor cell of pancreatic cancer cell strain T3M4 with high TrkB expression; b is a local enlargement of A, the triangle in the figure shows intercellular tight junction, AV is autophagosome, and the arrow shows autophagic vesicle.

FIG. 7 is an immunofluorescence map showing the transfer of gastric cancer cells highly expressing TrkB to mouse liver regions of the assembled organs; c and D are images after A and B are fused, wherein the green fluorescent protein marks gastric cancer cells which are up to TrkB.

FIG. 8 is an immunofluorescence map showing expression of enhanced blue fluorescent protein labeled LC3II in T3M4 pancreatic cancer metastatic large aggregates, A, C corresponding to different positions, B is an enlarged view of A, D is an enlarged view of C, and LC3 fluorescent spots in large aggregate cells can be seen (LC3 is a marker molecule for autophagy); e is the expression condition of the pancreatic cancer cell strain TrkB, wherein T3M4 is the highest.

FIG. 9 is a light microscopic image showing the interfhagy fusion (Interphagy) of peripheral cells of metastatic large aggregates of pancreatic cancer, continuously photographed kinetically over time.

FIG. 10 is a light microscopic photograph, A-O dynamic continuous photograph, from which it can be seen that the pancreatic cancer cell line T3M4 with siRNA inhibiting TrkB failed to form tumor cell aggregates in the anoikis state.

Fig. 11 shows the release of high-expression TrkB microvesicles surrounding large aggregates, where a is an immunofluorescence map, B is an electron micrograph, C is an immunohistochemistry map, and D is the selection of one microvesicle map C for immunohistochemical staining of TrkB cells.

FIG. 12 is a graph of immunofluorescence and immunohistochemistry showing the infiltration of pancreatic cancer cells highly expressing TrkB into the luminal lacunae of the pancreatic duct, which is the region of stem cells in which the pancreatic tissue itself is most actively dividing, B is an enlarged view of A, and D is an enlarged view of C.

Detailed Description

The research analysis and experiments and the like of the applicant are explained in detail below.

Tumor metastasis is a multistep, inefficient process. The impact of fluid mechanics in blood vessels and lymph vessels, hypoxia, nutrient deficiency, attack of the immune system, and uncomfortable tissue microenvironment all pose a fatal threat to metastatic tumor cells. Thus, applicants have recognized that distant metastasis of tumor cells is not done by individual cells alone, and that metastasis effectiveness is only ensured by aggregation to form large aggregates (typically > 50 cells) that form a mobile tumor microenvironment. Our previous studies found that TrkB of the nerve growth factor receptor family is a prerequisite for the maintenance of this large aggregate formation. For example, we find that TrkB can lead cytoskeletal protein beta-Catenin to be phosphorylated through activating a downstream Protein Kinase C (PKC) signal pathway and combine with E-cadherin protein on a cell membrane to achieve the effect that actin activity is involved in cell adhesion.

Large aggregates of tumor cells are exposed to various selective stresses from the peripheral environment during metastasis (e.g., fluid dynamics, immune system, anoikis, etc.), and we have recently discovered, pancreatic cancer cells highly expressing TrkB can achieve inhibition of anoikis through activation of PI3K/Akt/PKB/mTOR cell pathways (anoikis is a special form of apoptosis, and normal cells spontaneously die after leaving extracellular matrix in contact with the cells and neighbor cells around the cells due to the loss of the existing living environment, which is called anoikis, while cancer cells can still infiltrate and transfer far after leaving the original living environment, namely, the cancer cells can not generate anoikis, so that the defect of anoikis overcoming is a prerequisite for survival of the cancer cells in the process of infiltration and transfer). Moreover, tumor cells in the periphery of large aggregates can ensure the integrity of the aggregate structure through intercellular mutual phagocytosis (interphagy) and acquire necessary energy support through autophagy. It is thought that cell fusion is a means of generation of new genes and new traits evolved in nature, and that cell phagocytosis should be a self-defense manifestation of cells under selective stress. We have also observed that during the metastasis of large aggregates of tumour cells, there are always microvesicles (microvesicles) highly expressing TrkB released from the peripheral tumour cell membrane of the aggregates, which seem to be also an essential part of the protection of the metastasis of large aggregates of tumours, possibly associated with the inhibition of the immune surveillance and the immune escape of the organism on the tumour cells. This phenomenon is a heterogous isoworker with the recent findings that the microvesicles associated with melanoma cells and human colon cancer cells, which were first reported by lica rieoti, promote differentiation of human monocytes to myeloid-derived suppressor cells (MDSCs) and thus cause tumor immune escape. The above reports indicate that MDSCs can inhibit the acquired and natural anti-tumor immunity of the body through various ways, so that tumor cells can evade immune surveillance and attack of the body and promote tumor development. Therefore, it is suggested that these micro vesicles with high expression of TrkB around the large aggregates should also be a mechanism to protect the large aggregates from external shock during transfer.

Previous and recent studies demonstrated that high TrkB expressing tumors are more aggressive and less prognostic in breast, prostate, basal cell, lung, biliary, gastric, esophageal, hepatocellular, and pancreatic cancers. The invasiveness of tumor cells does not mean that they have a greater ability to metastasize clones. Interestingly, we found that tumor cells in the core of large aggregates of pancreatic cancer metastases appear to have stem cell-like characteristics that express molecular markers specific to stem cells. The characteristics of the stem cells should be exactly the reason why the metastasis of the pancreatic cancer large aggregates reach distant target organs such as liver and the like to be clonally planted and grown. From the perspective of biological evolution and genetic economy, the method for obtaining stem cells by tumor cells with the best and most rapid characteristics is to fuse with various precursor stem cells with stem cell characteristics to directly obtain the characteristics of the stem cells, i.e., the tumor cells with strong invasive ability obtain the characteristics of the stem cells and further have the ability of planting and cloning distant target organs. Recent studies have confirmed that bone marrow, the largest stem cell pool in humans, continuously releases various precursor stem cells to the periphery and has found their traces in some tumors, and we have newly found that pancreatic cancer cells highly expressing TrkB infiltrate into the caveolae of the pancreatic duct, which is just the stem cell region where the pancreatic tissue itself is most actively dividing with increased value. In 2008, Yamanaka et al successfully introduced Oct4, Sox2, c-Myc and Klf4 genes into liver and stomach Cells of experimental mice by using viruses, and Induced Pluripotent Stem Cells (iPS) from differentiated adult Cells, which is contrary to the hypothesis that tumors can obtain Stem cell characteristics by fusing with Stem Cells, provides another favorable support for our hypothesis. Tumor cells also appear to acquire stem cell characteristics by recruiting circulating stem cells for cell fusion through the synthetic secretion of various chemokines.

Therefore, we combined our previous and predecessor studies to derive such a possible hint: in pancreatic cancer, pancreatic cancer cells highly expressing TrkB are fused with stem cells migrated by bone marrow or pancreatic cancer cells highly expressing TrkB invade pancreatic tissues and are fused with stem cells of pancreas to obtain the characteristics of the stem cells, then large aggregates are formed to protect the fused tumor stem cells from being transferred to a distance, and the large tumor transfer aggregates are just like Trojan horses, so that the core tumor stem cells are protected from various selective pressures in the transfer process and the attack of an immune system, and the large tumor transfer aggregates can safely reach distant target organs to plant and clone to form a transfer focus.

Through the above studies, we can basically clarify the important roles of TrkB in the formation of tumor metastasis stem cells, metastasis large aggregates and maintenance of stability, and we expect to analyze the role of each node in the above TrkB-mediated fusion of relevant cells, formation of large aggregates and formation and release of microvesicles by starting with each signal node on 3 signal transduction pathways known to be associated with TrkB, and strive to find one or several optimal targets as the direction of treatment for inhibiting the formation of tumor metastasis large aggregates to block tumor distant metastasis (several optimal targets are isoaspected with cocktail therapy).

Brief description of relevant experimental (validation) procedures:

firstly, to explore the source of metastatic tumor stem cells of pancreatic cancer, whether highly invasive pancreatic cancer cells are fused with intraglandular stem cells or bone marrow circulating stem cells are recruited or both are verified. As shown in fig. 1 and 2.

In our previous experiments, human pancreatic cancer cell strain T3M4-GFP of Green Fluorescent Protein (GFP) and T3M4-GFP cell transfected by TrkB-siRNA have been successfully constructed. The observation of the fusion of T3M4 cells containing TrkB and inhibiting TrkB with pancreatic tissue in situ or circulating bone marrow cells explored the mechanism that TrkB gene's high invasiveness might promote cell fusion.

For verifying whether tumor cells in a large aggregate formed when pancreatic cancer cells highly expressing TrkB are transferred have the characteristics of stem cells, a three-dimensional collagen culture matrix is prepared by using type I collagen, the large aggregate is placed in the collagen culture matrix and is rapidly frozen into slices, and the position distribution of the tumor cells with the characteristics of the stem cells can be identified by using an immunofluorescence antibody from the longitudinal slices. The cells of positive expression stem cell characteristic molecular markers (CD24+, CD44+ and ESA +) in the aggregate core are cut by a laser micro-dissection technology, and then the Oct4, Oct3, Sox2, c-Myc, Nanog, Lin28 and Klf4 stem cell characteristic activity genes in the cells are detected and screened by RT-PCR, so that the aim is fulfilled. We tentatively define 2 or more surface molecule positive combined 1 transcription factor positive cells as tumor cells with stem cell characteristics, and the obtained cells are subjected to experience of stem cell activity of clone proliferation and metastasis in a nude mouse liver transplantation model.

For identifying whether the characteristics of the stem cells are obtained by fusing highly invasive pancreatic cancer cells with stem cells inside an glandular tube or recruiting bone marrow circulating stem cells or both, the method adopts male BALB/C-nu nude mouse bone marrow, transplants the bone marrow damage dose-optimized female BALB/C-nu nude mouse, transplants T3M4-GFP pancreas with high expression TrkB into the bone marrow damaged female nude mouse in situ, and judges whether the pancreatic cancer cells are fused with circulating bone marrow cells by detecting the existence of Y chromosome in the tumor stem cells through SRY chromosome fluorescence in situ hybridization and SRY gene (male sex determining gene, gene segment which specifically determines the male sex of an organism on Y chromosome) PCR.

Secondly, regulation and control of the structural stability of the metastatic large aggregate of the tumor cells can occur at various levels, including adhesion regulation and aggregation and coordination with a cytoskeleton structure, and previous researches prove that pancreatic cancer cells with high expression of TrkB can realize inhibition of anoikis through activation of a PI3K/Akt/PKB/mTOR cell pathway, so that survival of the metastatic tumor cells is promoted. As shown in fig. 3. In the subsequent research, the activities of each cell adhesion molecule signal channel and cytoskeleton molecule channel are respectively examined in a pancreatic cancer cell strain T3M4 with high expression TrkB and a pancreatic cancer cell CAPAN-1 with low expression TrkB under the condition of simulating the hemodynamics, and the dynamic changes of the signal channels are examined on T3M4 interfered by siRNA and CAPAN-1 cells transfected with TrkB.

Thirdly, the tumor cells escape from the monitoring and attacking of the host immune system, and a plurality of means are developed, including covering, modifying, mutating and deleting autoimmune markers to escape from the immune monitoring and recognition of the host, and on the other hand, the tumor cells achieve the aim of inhibiting the anti-tumor immunity of the organism by destroying the structure and the function of immune effector cells, so that the tumor obtains immune escape to promote the development of the tumor cells. In the research, firstly, fusion tumor stem cells which are obtained by the experiment of fig. 1 and highly express TrkB are used for continuously inducing the formation of large aggregates under the simulated environment of the castration fluid industry dynamics in RWV, then immunomagnetic beads (MACS) are used for enriching and separating TrkB microvesicles which circulate around the moving large aggregates to verify whether the TrkB microvesicles can induce the release of bone marrow-derived suppressor cells (MDSCs), and further an interferon release test is used for verifying whether the TrkB microvesicles can mediate the abnormity of T cell functions and whether the TrkB microvesicles can mediate the abnormity of antigen presenting cells (DC cells) functions, and the research is carried out at the angles that the TrkB microvesicles can mediate the abnormity of the functions of hamster in-situ pancreatic cancer hamster models which are established by people after being enriched, and the immune function change and the tumor metastasis condition of hamsters are observed. As shown in fig. 4.

The research results are as follows:

the systematic and intensive research on the effects of neurotrophic factors (NGF and BDNF) and receptors thereof (TrkA, TrkB and P75NTR) in pancreatic cancer infiltration and metastasis finds that the NGF and the receptors thereof TrkA/P75NTR are not only involved in development, survival and function maintenance of central and peripheral nerves, but also have the effects of inducing apoptosis and promoting division and survival of cancer cells in pancreatic cancer, the growth promotion effect of the NGF on pancreatic cancer is related to the expression level and the ratio of TrkA/P75NTR, different expressions of the NGF and the receptors thereof are related to pancreatic cancer nerve infiltration and metastasis, high-frequency infiltration and metastasis are often accompanied by high-level expression of the NGF and TrkA, and conversely, high expression of the P75NTR is related to good prognosis. We also found that PI3K/AKT pathway protein kinase activity was reduced in pancreatic cancer cell lines that highly express p75NTR compared to cell lines that less express p75NTR, and that activation of this channel is an important pathway for tumor cells to resist the onset of anoikis. Interestingly, our recent studies also found that the autophagy phenomenon was involved in the energy supply of metastatic tumor cells, see fig. 6. In FIG. 6, the triangles show tight junctions between cells, AV is an autophagosome, and the arrows show autophagic vesicles; b is a partial enlargement of A.

We adopt different methods to respectively prove that the expression of TrkB in gastric cancer is related to the differentiation degree of tissues from different horizontal angles, and the expression of TrkB in a high-differentiation tumor is obviously higher than that of a low-differentiation tumor. The expression of TrkB in gastric cancer is obviously related to distant metastasis of gastric cancer, particularly liver metastasis, and the fact that the expression of TrkB in a patient with high-differentiation gastric cancer can be used as a predictor of distant metastasis, particularly liver metastasis is shown in figure 7. As can be seen from FIG. 7, the high TrkB-expressing gastric cancer cells transferred to the murine liver manifold region, C and D are A, and B is a fused image, wherein the green fluorescent protein marks the high TrkB-expressing gastric cancer cells.

We induced the formation of large aggregates by pancreatic cancer cell line T3M4 highly expressing TrkB in the anoikis state, we observed that large aggregate cells can supply self-energy through autophagy phenomenon to achieve the effect of resisting various selective pressures in the metastasis process, as shown in fig. 8; and the interfhagic fusion phenomenon between peripheral tumor cells of the metastatic aggregates was observed, as shown in fig. 9. siRNA inhibited TrkB pancreatic cancer cell line T3M4 from failing to form tumor cell aggregates in an anoikis state, as shown in fig. 10. The metastatic large aggregates formed by pancreatic cancer cell strain T3M4 highly expressing TrkB in the nested state are released by a plurality of micro vesicles highly expressing TrkB and surround the large aggregates in the simulated vascular hydrodynamic transfer state, as shown in FIG. 11. We observed that pancreatic cancer cells highly expressing TrkB infiltrated the luminal crypt of the pancreatic duct, which is the region of stem cells where the pancreatic tissue itself proliferates most actively, as shown in fig. 12.

From fig. 8, the expression of enhanced blue fluorescent protein labeled LC3II in T3M4 pancreatic cancer metastasis large aggregates can be seen, and the fluorescent spot of LC3 in cells of the large aggregates can be seen (LC3 is a marker molecule for autophagy). E is the expression condition of the pancreatic cancer cell strain TrkB, wherein T3M4 is the highest.

The phenomenon of interfhagy fusion (Interphagy) of peripheral cells of metastatic large aggregates of pancreatic cancer can be seen in fig. 9; continuous dynamic photography of a-F over time.

As can be seen from FIG. 10, the pancreatic cancer cell line T3M4 with siRNA inhibiting TrkB failed to form tumor cell aggregates in the anoikis state, and A-O dynamic continuous photographs were taken.

As can be seen from fig. 11, the high TrkB expressing microvesicles were released and surrounded by large aggregates (a, B) and image D was a selection of microvesicle image C for TrkB cell immunohistochemical staining.

As can be seen from FIG. 12, pancreatic cancer cells highly expressing TrkB infiltrated the luminal crater of the pancreatic duct, which is the region of stem cells in which the pancreatic tissue itself proliferates and divides most actively.