CN106421788A - Application of VEGF-C in screening of medicine for diagnosing or treating implantation metastasis cancer - Google Patents

Abstract

The invention discloses application of VEGF-C in screening of medicine for diagnosing or treating implantation metastasis cancer, and belongs to the field of medicine. It is proved that TAM relevant spheroids and the ovarian cancer clinical pathology are closely connected, an ovarian epithelial cancer model established in a mouse indicates that TAM promotes spheroid forming and tumor growing in the ovarian cancer implantation metastasis early stage, after EGF excreted by TAM activates EGFR on tumor cells, the EGF up-regulates tumor cells VEGFs-VEGFRs pathway signal transduction in return and then promotes tumor cell proliferation and differentiation, the EGF can up-regulate expression of alphaMbeta2 integral protein on TAM and expression of ICAM-1 on the tumor cells, and the tumor cell-TAM association is promoted. After TAM or medicine retardant VEGF or ICAM-1 in antibodies are removed from the rat, spheroid forming and ovarian cancer progression are hindered. A new mechanism of TAM mediated spheroids is disclosed, and new targeted therapy is provided for ovarian cancer and other distant metastatic cancer.

Description

VEGF-C screening for diagnose or treat Implantation matastasis cancer medicine in purposes

Technical field

The present invention relates to VEGF-C screening for diagnose or treat Implantation matastasis cancer medicine in purposes, belong to medical science Field.

Background technology

Oophoroma (OC) is one of kinds of tumor and major causes of death that the whole America gynaecology is number two.High mortality is main It is because oophoroma is in early stage (I/II) difficult diagnosis, until its diffusion can be made a definite diagnosis after advancing to late period (III/IV).We Have been reported that diagnosis rate from I phase to IV for the OC patient and be respectively 7.19%, 8.63%, 72% and 72.18%.The prognosis of OC patient Bad, each phase OC patient five annual survival rate is 42%.Find after the long term follow-up of III phase and IV phase patient is observed its five Year, survival rate was less than 10%.But the patient making a definite diagnosis for early stage (I-II), particularly those tumours are confined to the trouble of primary part Person, its five year survival rate reaches 92.7%.Research display 5 years survival rates of OC patient rise less than 2% compared with before 10 years.Extensively abdomen In chamber and pelvic cavity, Implantation matastasis are the main causes of OC prognosis mala, and these focal cleanings generally can not be done by operative treatment completely Only.In this case, for most of OC patients, surgical cytoreduction becomes last selection.Up to the present still do not have , to Implantation matastasis curative effect, and the chemotherapeutics being currently used for OC easily induces medicine to be found effectively specific targeted drug Drug resistance and treat after patient long-term prognosis unsatisfactory, therefore, the mechanism understanding fully OC Implantation matastasis is necessary, its for The research and development for the treatment of OC Implantation matastasis targeting novel drugs and raising OC survival are all most important.

After peritoneal surface is broken in swollen thing increase, tumour cell splits away off from carcinoma in situ, carries out in abdominal cavity with ascites Until field planting is in abdominal cavity, this is the explanation at present peritoneal implantation metastasis phenomenon the most generally admitted for transhipment.Many researchs are recognized Process for Implantation matastasis is divided into several steps as follows：1) cell detachment, survival and opposing anoikis；2) protected from immune prison Depending on；3) epithelial-mesenchymal transdifferentiation；4) orbicule is formed；5) ascites formation and 6) peritoneal seeding.But, free dropping swollen The mechanism that how oncocyte survives in Implantation matastasis environment and Implantation matastasis initial stage spheroid is formed is still unclear.

Content of the invention

It is an object of the invention to disclose the related mechanism of OC Implantation matastasis by mouse original position Ovarian Cancer Model, to swollen Knurl associated macrophages (TAMs) act on played in OC Implantation matastasis certain new knowledge, consequently, it is possible to provide blocking tumour kind Plant the novel targets of transfer.

For achieving the above object, the technical scheme that the present invention takes is：VEGF-C is in screening for diagnosis or treatment plantation Purposes in the medicine of metastatic carcinoma.

As the improvement further to technique scheme, the Implantation matastasis of described Implantation matastasis cancer and macrophage phase Close.As the improvement further to technique scheme, described Implantation matastasis cancer is oophoroma, cancer of pancreas or colorectal cancer.

As the improvement further to technique scheme, described medicine passes through to reduce VEGF-C level, retardance VEGF-C Acceptor or blocking VEGF-C downstream signal treating Implantation matastasis cancer.

As the improvement further to technique scheme, the downstream signal of described blocking VEGF-C passes through to suppress ICAM-1 Come to carry out.

As the further improvement to technique scheme, described medicine passes through to reduce VEGF-C level or retardance The acceptor of VEGF-C and suppress EGFR positive tumor cell migration and tumour cell-TAMs spheroid formation come to treat plantation turn Move cancer.In addition, present invention also offers the downstream signal antagonism of the beta blocker of VEGF-C inhibitor, VEGF-C or VEGF-C Medicine is used for the purposes in the medicine treating Implantation matastasis cancer in preparation.

As the improvement further to technique scheme, described VEGF-C inhibitor is VEGF-C neutralizing antibody.

As the improvement further to technique scheme, the beta blocker of described VEGF-C is VEGFR3 antagonist MAZ51.

As the improvement further to technique scheme, the downstream signal antagonistic of described VEGF-C neutralizes for ICAM-1 Antibody.

It should be noted that the neutralizing antibody occurring in the present invention namely common antibody, why referred to as neutralizing antibody master To name from the treatment angle of antibody.

The beneficial effects of the present invention is：Present invention shows that TAMs in oophoroma Implantation matastasis orbicule forming process Play requisite effect.TAMs can secrete a large amount of EGF thus the EGFR, the EGF/ of activation that activate in surrounding tumor cells EGFR signal path can raise VEGF-C, and it raises integral protein and ICAM-1 in turn thus forming autocrine positive feedback loop again Road, and then promote tumor cell proliferation, differentiation, adhesion, form orbicule and peritonaeum transfer, disclose the key of orbicule formation Mechanism, provides a kind of New Policy and the prognosis situation improving OC patient for suppression Implantation matastasis.

Brief description

Fig. 1 shows that macrophage (TAMs) participates in the formation of spheroid in the original location in Ovarian Cancer Model；To stably express The ID8OCs of mCherry fluorescent protein labeling implants 8 weeks big tomato^lyz-creIn Recipient mice body, plant respectively in oncocyte Enter the Cherry that abdominal cavity is penetrated in detection when 2,4,6 and 8 weeks⁺Tumour cell and GFP⁺Cell；Wherein, A is that abdominal cavity cell is applied by display It is placed in the fluorogram of fluorescence microscopy Microscopic observation after section is upper, each time point takes n=5 mouse；B is to GFP⁺Cell is total Number quantified, illustration be to from 0 to 20 days to GFP⁺The statistical chart that TCS is quantified, each time point only takes n=5 Mouse；C is to Cherry⁺Tumour cell sum is quantified, and illustration is 0 to 20 days to Cherry⁺The tumour cell sum amount of carrying out The statistical chart changed, each time point takes n=5 mouse；D-G display macrophage and spheroid are formed, and typical case during 3 to 6 week is glimmering As shown atd, figure E is the quantitative statisticses figure to spheroid sum [spheroid/100 μ l ascites] for light image, and figure F is [thin to spheroid volume Born of the same parents' number/spheroid] quantitative statisticses figure, each time point takes n=5 mouse, has proven to when 3 weeks to start to start spheroid and is formed；G Be with APC (647nm)-anti-CD68 and DAPI of mating type to 8 weeks when the spheroid that gathers carry out after immunostaining with confocal imaging skill The image of art gained, to GFP⁺And CD68⁺Macrophage, Cherry⁺Tumour cell and DAPI carry out visible after nuclear targeting, Right figure show fused image, and all data results are represented with means standard deviation form, n=5, and asterisk * is P<0.05, star Number * * is P<0.01, asterisk * * * are P<0.001.

Fig. 2 shows that M2 type TAM (TAMs) polymerization is related to the progress of oophoroma；Original position oophoroma F4/80 in model⁺CD11b⁺Macrophage is derived from the individual cells group that (1,4 and 8 weeks) put at fixed time or spheroid；A is use The statistical chart that qRT-PCR technology is detected to the mark of M1 type specificity and M2 type specificity, with PMBC As comparison, compared with the gene expression in individual cells body in 1 week, all data results are with means standard deviation form table Show, n=5, asterisk * is P<0.05, asterisk * * are P<0.01, asterisk * * * are P<0.001 (sided t distribution inspection)；B-C is use The statistic analysis result to expression in macrophage for CD163 and CD206 in the 1st and 8 week individual cells group for the FACS method Figure, all data results are represented with means standard deviation form, and n=5, asterisk * are P<0.05, asterisk * * are P<0.01, star Number * * * is P<0.001.

Fig. 3 shows that abdominal cavity spheroid is formed and ovarian cancer tumor cell growth institute is required；Mouse ID8 cell is injected Orthotopic mouse OC model is set up, then mouse gives liposome (ID8 group) or chlorine phosphine respectively in C57BL/6 female receptor mouse peritoneal Acid disodium liposome (ID8+LC group) is treated, and half donor mice receives ID8 cell and adds from suffering from cancer donor mice spheroid The TAMs separating and liposome (ID8+TAM) or disodium clodronate liposome (ID8+TAM+LC)；A-D shows Clodronate The two sodium lipidosomes and TAM effect to tumour growth, A is the increase curve map of at the appointed time (0-60 days) Mouse Weight；B-C It is shown in the statistical results chart of measurement ascites volume and tumour net weight when the 60th day, data is represented with mean+SD, n= 10, asterisk * * * are P<0.001；D is the monitoring quantitative statisticses figure to its survival rate for the mouse state, and every group of n=24 mouse is used Logarithm rank test carries out Kaplan-Meier analysis, and asterisk * * * is P<0.001；E-G display disodium clodronate liposome and The influence that TAM is formed to spheroid, figure E is to collect spheroid when 8 weeks from ascites to carry out H＆E colored graph, and figure F is spheroid sum Statistical chart, figure G is spheroid size statistical chart；H-J display disodium clodronate liposome and TAM act on to growth of tumour cell, and H is When gathering 8 weeks, individual cells group and spheroid, are carried out after immunostaining with confocal imaging to it with anti-Ki67, anti-CD68 and DAPI The coloration result figure that technology is observed, H is shown in CD68 in ID8 group⁺Macrophage is by Ki67⁺Tumour cell surrounds, and in ID8+LC Group has no, engineer's scale is 25 μm；Figure I is Ki67 in the spheroid overall and total to individual cells group⁺Enter the statistical results chart of quantization； Figure J is CD68 in the spheroid overall and total to individual cells group⁺The statistical results chart that cell is quantified, n=5 mouse, often Mouse provides 10 spheroids, and all data results are represented with means standard deviation form, and asterisk * * * is P<0.001 (sided t Distribution inspection).

In Fig. 4 display TAMs, EGF is mutually raised with EGFR expression in tumour cell and oophoroma growth is played a key effect； A-C display TAMs promotes external ID8 cell proliferation, after tumour cell is implanted 8 weeks from OC- model mice body in gather ascites Spheroid.TAMs and ID8 tumour cell (PE-ID8) is separated, individually cultivates PE-ID8 cell or exist with TAMs Cultivate together in transwell but be not in direct contact with it, primary ID 8 cell is as comparison；A is at the appointed time TCS Statistical results chart；B is the coloration result figure after cell growth Ki67 dyeing, 25 μm of engineer's scale；C is to Ki67⁺Tumour is thin The statistical results chart that born of the same parents are quantified, n=9；D be qRT-PCR detection PE-ID8 and spheroid in EGF, FGF, PDGFs, HGF and The column statistical chart of the gene expression amount of VEGFs；E is the EGF in TAMs and the expression of the EGFR in PE-ID8 cell system Meter figure, with qRT-PCR to the EGF in TAMs and PE-ID8 group and EGFR gene expression detect, PMBC and Respectively as control group, related gene expression assumes multiple and increases primary ID 8 tumour cell, using monocyte number as 1.0, n =3；F is the colored graph that with EGF or EGFR, the spheroid CD68 cell being isolatable from OC mouse ascites is carried out with immunostaining, n=5 The typical image of its spheroid of mouse is as shown in the figure；G is the EGF expressing quantity of ELISA detection 4 weeks and 8 weeks ascites supernatants Column statistical chart, blood plasma is as negative control；H detects what tumour cell, spheroid or tumour cell ball adding body co-cultured for ELISA The column statistical chart of EGF protein expression level in culture medium supernatant；I-J makes EGF silence express by siRNAs, and TAMs is by EGF SiRNAs or reference siRNA transfects 48 hours, and I is the statistical results chart detecting EGF mRNA level in-site in TAMs through qRT-PCR method； J is the statistical results chart detecting the EGF protein level in TAMs and ID8 co-culture of cells system supernatant with ELISA method；K-L Display processes ID8 cell 12 hours in no EGFR antagonist and in the case of there is antagonist (10 or 20 μM) with EGF respectively, with Corresponding antibodies are detected with total EGFR and ERK1/2 to phosphorylation by immunoblotting, total EGFR, ERK1/2 All detected with GAPDH, Relative phosphorylation level is quantified, K is Western blotting result figure, L is statistics knot Fruit is schemed；M-N display TAMs, with having carried out pre- transfection with reference to siRNA or EGFsiRNA, PE-ID8 cell individually or with TAMs is existed Carry out co-culturing 12 hours in the case of absence or presence EGF (20ng/ml) or EGFR antagonist (20 μM), figure M is to use Ki67 The coloration result figure that the PE-ID8 cell of hyperplasia is dyeed, 100 μm of engineer's scale；Figure N quantifies to Ki67 cell Statistical results chart, all tests have all carried out three groups of independent experiments, and data to be represented with average ± SEM, and asterisk * * is P< 0.01, asterisk * * * are P<0.001.

Fig. 5 shows the clinical correlation between OC patient EGF+TAMs and EGFR+ tumour cell and spheroid formation；A be to from The TIS separated in OC patient and spheroid carry out the coloration result figure that H＆E and CD68IHC dyes, and engineer's scale is respectively 50 μm (H＆E) and 25 μm (CD68)；B is the statistic analysis result to CD68 positive cell in OC patient's TIS and spheroid Figure, n=128, data mean+SD represents, asterisk * * * is P<0.001 (sided t distribution inspection)；C-D shows huge The correlation that phagocyte and cancer cell grow in spheroid；C be respectively to people little, neutralization large volume OC spheroid in CD68 and Ki67 carries out the coloration result figure of immunostaining, and wherein DAPI is used for nuclear targeting, 5 μm of engineer's scale；D is to CD68 in spheroid The statistical results chart being quantified with Ki67 positive cell, n=30, spherula (0-50 cell/spheroid), intermediate cell cluster (50-500 cell/spheroid), big spheroid (>=500 cell/spheroids), data is represented with mean+SD, asterisk * * For P<0.01, asterisk * * * are P<0.001, small size spheroid with, P compared with large volume spheroid<0.001 (sided t distribution inspection Test)；E is the fluorescent staining figure that the spheroid in OC patient's ascites is carried out with immunofluorescence dyeing, is joined using EGF or EGFR and CD68 Close dyeing, from n=128 example OC patient's spheroid typical image as illustrated, arrow pointed location be spheroid central authorities CD68+ EGF+TAMs, engineer's scale, 10 μm；F is the statistical results chart of its CD68+ cell in the OC spheroid of different differentiation degrees；G is Low in 128 OC patient's spheroids (<14.5%) or high (>=14.5%) CD68⁺Cell percentages and overall survival rate Kaplan- Graph of a relation (being analyzed with logarithm rank test) between Meier curve.

Fig. 6 is shown in mouse model and blocks EGFR minimizing spheroid formation, cell proliferation and oophoroma growth；By to female Property acceptor nude mice abdominal cavity in injection people SKOV3OCs set up orthotopic mouse model, then mouse is not processed with (CON) or gives Tarceva is treated, and LC is as treatment control group；A-E shows the impact to SKOV3 tumour growth for the Tarceva；A be Corresponding time point monitors the statistical results chart of Mouse Weight, arrow indication be using LC treatment the different initial treatment times ( When tumour cell is implanted 2,4 or 8 weeks)；B is the statistical results chart monitoring Mouse Weight in corresponding time point, and arrow indication is to make The different initial treatment times (when tumour cell is implanted 2,4 or 8 weeks) with Tarceva treatment；C is CON, LC and Lip river in distress is replaced The whole body representative pictures of Buddhist nun treatment group mouse；D is the statistical results chart weighing mouse ascites volume when 14 weeks；E is to claim when 14 weeks The statistical results chart of amount mouse tumor net weight；In wherein A-E, data is represented with mean+SD, n=10 in every group, star Number * * * is P<0.001；ns:There was no significant difference；F combines overall survival rate Kaplan-Meier of control group for Tarceva Graph of a relation between curve；G-I shows Tarceva effect that SKOV3 spheroid is formed, G be spheroid in collection ascites when 14 weeks simultaneously It is placed on smear, spheroid is carried out with the coloration result figure of H＆E dyeing, 100 μm of engineer's scale；H quantifies to spheroid sum Statistical results chart；I is the statistical results chart that spheroid volume is quantified；J-K display Tarceva increases to SKOV3 tumour cell The effect grown, the spheroid gathering during to 14 weeks carries out immunostaining with anti-Ki67, anti-CD68 and DAPI, then with confocal imaging Technology is observed；J is containing Ki67⁺Tumour cell and CD68⁺The immunofluorescence dyeing figure of macrophage spheroid；K is to spheroid Interior Ki67⁺And CD68⁺The statistical results chart that cell is quantified, n=5 mouse, gather 10 spheroids, data in every mouse Represented with mean+SD, asterisk * * * is P<0.001.

Fig. 7 shows that TAMs passes through to secrete the migration that EGF promotes tumour cell；A-B is ID8 cell in transwell cell Individually cultivate, or co-culture with mouse monokaryon cell or with TAMs, with the transfer ability of scratch experiment quantitative analysis ID8 cell, A For the representative picture of tumor cell migration, B is statistic analysis result figure, n=3,100 μm of scale；C-D exists for ID8 cell Transwell cell is individually cultivated, or co-cultures with mouse monokaryon cell or with TAMs, counts through transwell cell Cell number carrys out the transfer ability of quantitative analysis ID8 cell, and C is to carry out haematoxylin to the tumour cell through transwell cell The colored graph of dyeing, D is the statistic analysis result figure of cell migration number, n=3,50 μm of scale；E-F is that the pre- transfection of TAMs is right After siRNA or EGF siRNAs 48 hours, and then the TAMs cell of ID8 cell and transfection is trained altogether in transwell cell Support, in PE-ID8 culture liquid system plus or be not added with EGF (20ng/ml) or EGFR inhibitor Tarceva (20 μM), E is to wearing The tumour cell crossing transwell cell carries out the coloration result figure of haematoxylin dyeing, and F is statistic analysis result figure, scale 100 μm, data is represented with mean value ± standard error, n=3；Asterisk * * * is P<0.001 (double tail t inspection).

Fig. 8 is shown in EGF in oophoroma macrophage and raises VEGF-C-VEGFR3 signal, and A-B is real-time quantitative qRT- PCR detects the mRNA expression of VEGF-C and VEGFR3 detached TAMs and PE-ID8 tumour cell in mouse body, and A is The mRNA relative expression quantity statistical results chart of VEGF-C, B is the mRNA relative expression quantity statistical results chart of VEGFR3, thin with monokaryon The mRNA expression of born of the same parents is 1, the multiple change of the relative mRNA expression levels of VEGF-C and VEGFR3 of other group cells, n =3；C detects the albumen of VEGF-C, the VEGFR3 of phosphorylation and total VEGFR3 in ID8 and PE-ID8 cell for immune protein trace The Western blot figure of level, lymph vessels endothelial cell (LECs) conduct that will process with or without VEGF-C (20ng/ml) Positive control；D is the column diagram of the albumen relative expression quantity of VEGFR3 of quantitative analysis VEGF-C and phosphorylation；E-F is mouse ID8 cell and people's SKOV3 ovarian cancer cell process different time (0-12h) with EGF (20ng/ml), use real-time quantitative qRT-PCR Detect the column diagram of two groups of VEGF-C mrna expression amounts, with the VEGF-C mrna expression amount of untreated fish group for 1, other times point VEGF-C mrna expression amount changes display with multiple；G-H is to process ID8 cell with EGF during different time points, and G is to VEGF- The Western blot figure that C, the VEGFR3 of phosphorylation and total VEGFR3 protein level are detected, H is to VEGF-C and phosphorylation The statistical results chart that the protein versus expression of VEGFR3 is quantified；I is short of money in EGFR antagonist (20uM) or ERK respectively In the case that anti-agent (PD98059,20uM) adds and lacks, with EGF, SKOV3 cell is carried out with the process of 12 hours, I is use The statistical results chart that qRT-PCR method is detected to VEGF-C mRNA, if it was found that using untreated fish group result as 1.0, the VEGF-C of EGFR antagonist or ERK antagonist group is calculated relative to mRNA level in-site (with GADPH for standard with untreated fish group As internal reference) multiple change；J is to exist in EGFR antagonist Tarceva (20uM) respectively and use EGF in the case of lacking ID8 cell is processed 12 hours, the Western blot figure of the VEGFR3 to phosphorylation and total VEGFR3 albumen, with VEGF-C process (concentration is 20ng/ml, 5 minutes time), as comparison, all data are represented with mean value ± standard error, and n=3, asterisk * * * are P< 0.001 (sided t inspection).

Fig. 9 display EGF promotes EGFR+ tumor cell migration by autocrine VEGF-C/VEGFR3 signal pathway；A is right VEGF-C or VEGFR3 in OC mouse ascites spheroid and CD68 carries out the coloration result figure of immunofluorescence dyeing, from n=5 only The spheroid typical image of mouse；B-C display PE-ID8 cell is being added without or is adding respectively with EGF or VEGF-C (20ng/ml) Carry out the transwell migration of 12 hours in the case of EGFR antagonist Tarceva or VEGFR3 antagonist MAZ51 (10nM) Experiment；B is the coloration result figure using haematoxylin dyeing, engineer's scale 150um；C is the statistical results chart of Cell migration assay；D- F shows from lotus knurl tomato^lyzMThe mouse GFP isolating in-cre mouse spheroid⁺F4/80⁺CD206⁺TAMs and reference-siRNA Or EGF-siRNA carries out the pre- transfection of 48 hours, then TAMs and ID8 cell is existed in Tarceva or MAZ51 (20nM) In the case of carry out Combined culture in 3D co-culture system, D is fluorescence results figure, and engineer's scale 50um is it is seen that local GFP⁺Carefully Born of the same parents (TAMs) are located at control group spheroid central authorities but are not found in siEGF group, and E is after 48 hours, number of spheres (every hole) to be quantified Statistical results chart, F is statistical results chart that spheroid size (area) is quantified after 48 hours；G-I shows from people's ovary The CD14 that cancer patient's spheroid is isolated⁺TAMs has carried out the pre- transfection of 48 hours with reference to-siRNA or EGF-siRNA, for People's OC SKOV3 cell carries out 3D co-cultivation, and G is fluorescence results figure, and engineer's scale is 25um；H is that the number of spheres to 48 hours enters The statistical results chart that row quantifies；I is the statistical results chart that the spheroid volume to 48 hours is quantified, and all tests are all carried out Three independent experiments, data record in the form of mean+SD, asterisk * * * is P<0.001.

Figure 10 shows the mRNA expression in TAMs for CD11a, CD11b, the CD11c and ITG β 2.At the 8th week from spheroid Isolate TAMs the and PE-ID8 cell of infiltration growth Ovarian Cancer Model, A is to detect TAMs and PE-ID8 cell with qRT-PCR In the mRNA expression of CD11a, CD11b, CD11c and ITG β 2 integral protein statistical results chart, with primary ID 8 cell As comparison, all data are represented in the form of mean value ± standard error, n=5, and asterisk * * is P<0.01, asterisk * * * are P< 0.001 (sided t inspection)；B is the result figure CD11b+ and CD11c+ macrophage in abdominal cavity being analyzed with FACS technology, C is the statistical results chart respectively CD11b+ and CD11c+ cell being counted with percentage %, n=5, and asterisk * * * is P< 0.001, ns：There was no significant difference (sided t inspection), and in triplicate, data is represented with mean value ± standard error, star for all experiments Number * * is P<0.01, asterisk * * * are P<0.001.

Figure 11 display EGF/EGFR-VEGF-C/VEGFR3 signal path induction spheroid ICAM-1 expression, at the appointed time (8 When all) isolate TAMs the and PE-ID8 cell that infiltration grows Ovarian Cancer Model from spheroid, A is to detect ICAM- with qRT-PCR The statistical results chart of 1 and ICAM-2 mRNA expression, using PMBC as comparison, all data are with mean value ± standard error represents, n=5, and asterisk * * is P<0.01 (sided t inspection)；B is to process ID8 cell different time with EGF, uses The statistical results chart of the mRNA expression of qRT-PCR technical Analysis ICAM-1, carries out three independent repeated trials, and data is with flat Mean value ± standard error represents, asterisk * * is P<0.01, asterisk * * * are P<0.001 (sided t inspection)；C-D is thin to ID8 with EGF Born of the same parents process different time, and C is the Western blot figure of the protein expression detecting ICAM-1 with western blotting method, and D is quantitative statisticses The column diagram of the protein expression level of analysis ICAM-1；E-F is with VEGF-C, ID8 cell to be carried out processing different time, and E is use Western blotting method detects the western blot figure of the protein expression of ICAM-1, and F is the protein expression of quantitative statistical analysis ICAM-1 The column diagram of level, in triplicate, with mean value ± standard error, n=3, asterisk * * * are P to data for all experiments<0.001 (bilateral T checks)；G is respectively in the case of adding VEGFR3 antagonist (MAZ51) and its disappearance, thin to ID8 with EGF or VEGF-C Born of the same parents carry out the process of 24 hours, and with qRT-PCR method, the mRNA expression of ICAM-1 is detected and carried out with the post of quantitative analysis Shape figure, in triplicate, data is represented with mean value ± standard error for all experiments, and asterisk * * is P<0.01, asterisk * * * are P< 0.001 (sided t inspection).

Figure 12 display TAMs is passed through integral protein α M β 2 and ICAM-1 interaction and promotes to be sticked with EGFR+ tumour cell phase Attached；A is the coloration result figure that CD68 is carried out with the ICAM-1 extracting in OC mouse ascites spheroid with immunofluorescence dyeing, from n =5；B-C display PE-ID8 cell is with EGF respectively in absence or presence EGFR antagonist Tarceva or ERK antagonist Process 24 hours in the case of PD98059 (10nM), B is the Western blotting detecting ICAM-1 protein expression with Western blot Figure, C is the statistical results chart that ICAM-1 relative amount is quantified；D-E display ID8 cell is existed respectively with EGF or VEGF-C Process 24 hours in the case of absence or presence MAZ51 (VEGFR3 antagonist), D is to detect ICAM-1 albumen with Western blot The western blot figure of expression, E is the statistical results chart that ICAM-1 relative amount is quantified；In F-K display ICAM-1 and anti- The impact that body is formed to spheroid, F-H carries out 3D co-cultivation in the case of adding anti-mouse ICAM-1 to mouse TAM ID8, and F is to exempt from Epidemic disease fluorescence results figure, 50 μm of engineer's scale；G is the statistical results chart detecting number of spheres after 48 hours；H is to detect ball after 48 hours The statistical results chart of body size；I-K carries out 3D co-cultivation in the case of adding anti-human ICAM-1 to people TAM-SKOV3, and I is immunity Fluorescence results figure, 25 μm of engineer's scale；J is the statistical results chart of the quantity detecting spheroid after 48 hours；K is to detect ball after 48 hours The statistical results chart of the size of body, statistics all carries out 3 independent experiments, and data is represented with mean value ± SEM, and asterisk * * is P<0.01, asterisk * * * are P<0.001；L-Q shows by injection mouse ID8OCs in C57BL/6 female receptor mouse peritoneal Cell and set up orthotopic mouse model, then mouse is given Intraperitoneal injection IgG or anti-mouse ICAM-1 Antybody therapy, L be refer to (0-50 days) measurement Mouse Weights of fixing time increase the curve map of situation；M is at the appointed time to measure mouse ascites within (0-50 days) The statistical results chart of volume；N is the statistical results chart of at the appointed time (0-50 days) measurement mouse tumor weight；O is to spheroid Carry out the coloration result figure of H＆E dyeing；P is the statistical results chart that the sum to spheroid is quantified；Q is that the size to spheroid is entered The statistical results chart that row quantifies, data is represented with mean value ± SEM form, and n=10, asterisk * * * are P<0.001.

Figure 13 display spheroid forms middle TAM- tumour cell interaction model, and A is EGF, VEGF-C, ICAM-1, α M β 2 Interact the schematic diagram adjusting, and is shown in the early stage of OC Implantation matastasis and tumour growth, and the OC cell induction coming off is huge to be bitten carefully Born of the same parents infiltrate in abdominal cavity, and under abdominal cavity environment, inducing macrophage is simultaneously for macrophage and OC cell interaction formation spheroid M2 hypotype TAM (TAMs), TAMs perhaps provide initial positioned at spheroid central authorities for OC escape anoikis Matrix supports it is important that TAMs can secrete a large amount of EGF thus activating EGFR, this can raise the expression of tumour cell, activation EGF/EGFR signal path can induce VEGF-C to express, and it activates VEGFR3 signal transduction pathway and induced tumor cell in turn The expression of interior integral protein/ICAM-1 thus forming a positive autocrine feedback control loop, thus promote tumor migration, adhesion and Spheroid is formed；B is the schematic diagram of TAMs and OC cellular elements interphase interaction.

Specific embodiment

For better illustrating the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiment and accompanying drawing pair The present invention is described further.

Embodiment

1. materials and methods

1.1 research approvals

All zooperies are carried out by after the approval of mechanism of the care of animal committee of Yale University.From informed consent, patient carries For ascites in gather late period (i.e. III-IV phase) ovarian cancer patients human tumour spheroid, be more than completed in Yale University's medical science Institute, New Haven, the Connecticut State (HIC agreement #1111003959).Clinical paraffin specimen is attached by Chinese Harbin Medical University Use after tumour hospital's Institutional Review Board approval.

1.2 animal model

All zooperies are implemented by after the approval of the care of animal committee of Yale University.All of mouse is C57BL/ 6 series.Lysozyme C re mouse and tomato (mT/mG) mouse are hybridized to be used lineage marker lysozyme to extract cell.Naked Mouse is purchased from Jackson laboratory.By 100ul mouse ovarian cancer ID8 on cell culture medium or HOC's SKOV3 cell (1 × 10⁶/ ml) it is injected in C57BL/6 system mouse or the abdominal cavity of nude mice.Mouse Weight increases, ascites volume and tumor weight change Situation is by electronic balance weighing.By sacrifice after 9 weeks, tumour spheroid and tumour transplatation thing carry out histologic analysis.

1.3 clinical sample

Through credentials committee approval, in January, 2005 in December, 2009 is in Harbin Medical University attached tumour doctor Totally 128 Advanced Epithelial Ovarian Cancer (EOC) patients that institute goes to a doctor wherein meet our inclusive criterias enter to organize our research Object.Include criterion of acceptability and include following several respects:(1) pathologic finding turns out to be III phase EOC；(2) there is complete clinic substantially Data；(3) cancer is not yet through any treatment；(4) severe complication or other malignant diseases do not occur；(5) inform patient and Its family members' state of an illness and before the treatment signature Informed Consent Form.All patients have all carried out complete surgical cytoreduction treatment. The tumour spheroid of late period (i.e. III-IV phase) ovarian cancer patients collects (HIC association by voluntary form from patient's ascites View #1111003959).

1.4 cell culture

ID8 (mouse ovarian cancer epithelial cell strain) is derived from Drs.Jack Lawler and bass Israel of Harvard Medical School female The Carmelo Nucera of executive officer's medical centre gives, and SKOV3 (people's ovary adenocarcinoma cells strain) obtains from ATCC.SK-OV-3 Diphtheria toxin is included to TNF and several cell toxicity medicament, cis-platinum and adriamycin etc. have resistance.ID8 and SKOV3 cell is in Dulbecco's improvement eagle culture medium (DMEM) (U.S. adds Li Fuliya, Life Technologies Corporation) Culture, wherein adds the streptomysin of 10% calf serum (FBS), 100U/mL penicillin and 100 μ g/mL, keeping temperature 37 DEG C it is placed in 5%CO₂In the moist environment mixing with 95% air.Change a subculture every 2 days, thin as 80-90% Born of the same parents occur to be isolated when merging.In all tests, cell is planted with rational density and is reached 80- before experiment proceeds by 90% cell fusion.C57BL/6 system mouse gives Intraperitoneal injection ID8 cell in different time points, hereafter divides from its abdominal cavity From the TAM (TAMs) obtaining Primary mouse and PE-ID8 cell.

1.5 statistical method

Western-blot, qRT-PCR, cell proliferation experiment, immunostaining, FACS and tumour growth are all examined using T Test analysis.By Chi-square Test or Fisher ' s, accurately inspection carries out statistical analysis to OC patient population factor difference.Kaplan- Meier method is used for assessing OS, meanwhile, corrects difference that may be present between Prognostic Factors with the logarithm rank test of single-factor analysis therapy Different.Multivariate Cox Regression (proportion grading model) is used for determining prognosis and independent factor CD68 level to OS.This research uses SAS software carries out statistical analysis (9.1.4 version, SAS software study institute, North Carolina State, New Caledonia).All Statistical check all adopts two-sided test to think statistically significant with P value less than 0.05 simultaneously.

1.6 recombinant proteins and antagonist

Recombined human and mouse EGF, VEGF-C purchase from R＆D Systems.MAZ51 (VEGFR3 antagonist) buys in EMD Millipore company.E Nuo replaces Buddhist nun's (EGFR antagonist) to buy from Roche company.For removing the Neutral Chloride fluorine of macrophage Loose disodium clodronate liposome is purchased from FormuMax Scientific company (product code #:F70101-N).

1.7 immunostaining

Antibody for SABC and immunofluorescence dyeing is listed in table one.Confocal images are by Zeiss- LSM700 microscope photographing is simultaneously processed by ZEN2010 software.For the measurement of average fluorescent strength, Laser Scanning Confocal Microscope figure As analysis is responsible for by ImageJ software.Cell section Zeiss Axiovert200 fluorescence microscope (the miniature one-tenth of karr Zeiss Picture；Sohne Wood, New York), image catches (Improvision, Lexington, Massachusetts) by Openlab3 software.? Frost, the tissue of OCT embedding are fixed 10 minutes, are dried with acetone under the conditions of being placed on -20 DEG C after carrying out 5 μm of serial section 15 minutes.

Table one antibody information sheet

1.8 protein extraction and western blot analysis

Loose being organized in lysis buffer of fresh cutting is homogenized, pyrolysis product is in 4 DEG C, 13,000g condition Under, be centrifuged 10 minutes, collect supernatant with Bradford determination of protein concentration kit (Bio-Rad company, Heracles, Plus Li Fuliya state) titrated.Cell lysate is analyzed with SDS-PAGE, then with Diagnosis of Sghistosomiasis together with specific antibody Mark method processes (Immobilon P；Millipore company, Penelope Milford, Massachusetts), finally tried with enhanced chemiluminescence Agent box (Amersham Life Sciences, Arlington highland, Illinois) is detected.All for protein immunoblot The antibody of method is all listed in Table 1.

1.9 fluorescence activated cell isolation technics (FACS)

By facs analysis mouse cell surface C D11b, F4/80, CD3e, CD206 and CD163 and human cell surface The expression of CD14 and CD326, existing document report before this.By the TAMs supensoid agent of mouse and mouse CD11b-FITC, F4/80- PE, CD3e-APC, PE-CD206, APC-CD163 and people CD14, CD326 antibody briefly mixes 15 minutes on ice.Of the same race anti- Body is as negative control.Flow cytometry is completed with FACSCalibur (BD life science).Data BD CellQuestPro Software is analyzed.All antibody for FACS are listed in table one.

1.10 migration experiment

Using transwell in vitro cell culture system (Corning company, aperture size, 0.3 or 8 μm) to huge bite thin The transfer ability of born of the same parents and tumour cell is detected, the driveization in order to complete ID8 cell migrates experiment, by 1x10⁴ID8 cell connects Plant in transwell chamber surface.Different chemotactic things (VEGFC or EGF) or macrophage (TAM) are placed under cell Orientation is put.After 12 hour incubation period, wipe the ID8 cell on transwell film upper strata away with cotton swab, in transwell film bottom surface Cell be fixed with 4%PFA after with Hematoxylin-eosin dyeing, under microscope (200X), then randomly select 5 visuals field Carry out the counting of migrating cell.

1.11 quantitative polyase chain reactions (qRT-PCR)

All RNA extracting from human body use RNeasy Plus Mini Kit (74134, Qiagen company), then High Capacity cDNA reverse transcription reagent box (4368814, Applied is used under manufacturer's instructions direct Biosystems company) it is transformed into cDNAs.Quantitative PCR uses RT2SYBR Green (330500, SA Biosciences) kit and CFX-96 (Bio-Rad) instrument complete.The all primers used in qRT-PCR are all in table two List.All of value is all standardized with the abundance of GAPDH.Data is represented in the form of three cell means ± standard deviation.

Table two primer information table

1.12TAM and ID8 cell 3D co-culture system

Mouse F4/80⁺CD206⁺TAMs passes through FACS from suffering from cancer donor tomato^lyzM-creIn the spheroid of mouse sorting and Come, collect after the human tumour spheroid of late period (i.e. III-IV phase) ovarian cancer patients (IHC agreement #1111003959), then use FACS identifies and sorts out CD14+TAMs.As mentioned above in 24 orifice plate upper berth matrigels, (ratio is 1 to TAMs and ID8cells: 10 but TCS is fixed as 40,000 cells/well) mixture be directly seeded on 24 orifice plates being paved with matrigel.Cell exists At 37 DEG C, culture ensures the formation of polymer/spheroid for 48 hours.Co-culture 6 hours after add EGFR antagonist Tarceva, VEGFR3 antagonist MAZ51 (each 20nM) or anti-ICAM-1 antibody (20ug/ml).Shoot fluorescence microscopy figure from 6-48 hour As carrying out morphological analysis.The aperture not having cell contains culture medium as the negative control of experiment.All experiments at least do 3 times And often secondary do three parts.Using Zeiss Axiovert 200 fluorescence microscope, (karr Zeiss micro is imaged；Sohne Wood, New York) Observe all MIcrosope images, image catches (Improvision, Lexington, Massachusetts) by Openlab3 software.

2. conclusion

2.1 macrophages participate in spheroid in oophoroma growth course and are formed

In order to determine whether macrophage participates in survival, propagation and the field planting of OC tumour cell during Implantation matastasis Etc. link, the applicant injects in C57BL/6 female receptor mouse peritoneal mouse ID8 cell thus setting up orthotopic mouse OC mould Type；For the Monocytes/Macrophages situation of cancer cell and Recipient mice in this period of tracing study, by ID8OC cell with steady The mCherry fluorescin of fixed expression is marked, meanwhile, using the tomato as Recipient mice^lyzM-creIn mouse body Myelocyte including macrophage is marked (GFP) with green fluorescent protein.

Result, as shown in the A-B of Fig. 1, is under base state (before tumour cell injection) or injects in tumour cell early In time period phase (<1 week) acceptor tomato^lyzM-creGFP can hardly be detected in mouse peritoneal⁺Cell, but, in injection Intraabdominal GFP is penetrated into when the after oncocyte the 2nd, 4,6 and 8 weeks⁺Cell has and is significantly increased, the GFP when 2,4,6 and 8 weeks⁺Carefully Born of the same parents' sum is respectively 3 × 10⁶、16×10⁶、18×10⁶And 20 × 10⁶.

As shown in C-D in Fig. 1, we also late detect CD11b in (6 weeks) ascites⁺Gr1⁺Marrow source property suppression cell (MDSCs) quantity increased, and in this group original position Ovarian Cancer Model, the cancer cell of injection initially enters rest period (0-2 week) Subsequently enter the rapid growth phase (when cancer cell injection 2-8 is all) (as Fig. 1 C), exactly like the II-III phase of HOC.Interesting It is, it is observed that having reduced to intraperitoneal tumor cell quantity in 2 time-of-weeks for initial 2 hours in injection oncocyte, can Can be for caused by anoikis.

As shown in D-F in Fig. 1, basis of microscopic observation finds that spheroid comprises GFP⁺Macrophage and mCherry tumour cell, The two ratio is 1:10；Number of spheres is increased with tumour growth with volume.It is interesting that as shown in Figure 1 G, spheroid tangent plane is exempted from Epidemic disease dyeing shows that the mCherry tumour cell in big spheroid is centered around the GFP positioned at ball centre position⁺CD68⁺Macrophage week Enclose, in CD11b⁺Gr1⁺Same result is obtained in MDSCs.

Result above all points out myeloid cell and the interaction of ID8 cell to promote spheroid during oophoroma Implantation matastasis Formation.

2.2 M2 type TAM (TAMs) polymerization is related to the progress of oophoroma

After injecting oncocyte 2 hours in mouse body, we are found that macrophage in its abdominal cavity, but macrophage Seem to enter, in oncocyte, the growth that tumour cell could be promoted after body forms macrophage-tumour spheroid in 3 weeks, have report before this Road in vivo and in vitro under environment ovarian cancer cell all macrophage is polarized to M2 type, it is presumed that macrophage forms the phase in spheroid There are different gene expression profiles and phenotype compared with initial period.In order to prove this point, we gather oophoroma difference growth step The F4/80 of section (when oncocyte injects 1,4 and 8 weeks)⁺CD11b⁺Macrophage, detects one group of M1 type-special mark by qRT-PCR Will thing and M2 type-specific markers, by PMBC as a control group.

Result shows infiltrating macrophages (when 1 week) the great expression M1 sample mark initially being produced by tumor cell induction Gene (Ly6G/C, CCR2, IFN α R, iNOS), but these marks are vanished from sight after 4 weeks.But, infiltrating macrophages During tumour progression, (when 4-8 is all) gradually obtains expression M2 phenotypic marker gene [CD206 (mannose receptor), CX3CR1, essence Propylhomoserin enzyme 1 and CD163 (scavenger receptor is rich in cysteine 1 type protein M130)] ability.Obviously, as shown in Figure 2 A, no By being single or spheroid correlation macrophage all has similar gene expression profile in latter stage (when 8 weeks)；As Fig. 2 B, 2C institute Show, facs analysis determine early stage CCR2⁺With late period CD163⁺,CD206⁺And CX3CR1⁺The macrophage of tumor inducing is different Expression way.

Result above shows that TAMs is polarized to M2 hypotype under abdominal cavity microenvironment during ovarian cancer progress.

2.3 TAMs is formed for abdominal cavity spheroid and ovarian cancer tumor cell growth is required

In order to inquire into TAMs role during oophoroma Implantation matastasis, we use disodium clodronate liposome to lotus Mouse is processed knurl.As shown in figures 3 a-c, phenotype analytical display disodium clodronate liposome significantly reduces Mouse Weight, ascites Volume and the tumour cell weight in wet base separated from ascites, therefore, through clodronate treatment mouse survival rate significantly Improve (Fig. 3 D), especially the number of spheres of clodronate group and mean size all significantly reduce compared with liposome group (Fig. 3, E-G), further study showed that Ki67 in spheroid⁺It is placed around CD68⁺Tumour cell around TAMs, through clodronate treatment CD68 afterwards⁺TAMs and Ki67⁺Cell quantity is all reduced (Fig. 3, H-J).

And then can we promote spheroid formation and ovarian cancer progress to carry out direct detection to TAMs.For achieving the goal, The F4/80 isolating in cancer mouse spheroid will be suffered from from donor⁺CD206⁺M2TAMs(1×10⁶) and ID8 cell (1 × 10⁶) together Inject in new Recipient mice abdominal cavity, Recipient mice, with liposome or chlorine disodium hydrogen phosphate liposome therapeutic, injects TAMs or ID8 thin Born of the same parents are as a control group.It is separately injected into TAMs and does not cause tumour, the detached TAMs cell for purely being polluted by ID8 is described. TAMs⁺Compared with ID8 group, the former significantly increases gross tumor volume, increases ascites and tumour weight in wet base ID8 group.But, Clodronate two Sodium lipidosome treatment group and TAM⁺ID8 Comparatively speaking, the increase of the former body weight, ascites and tumor quality not as good as the latter, or even Less than ID8 group (Fig. 3, A-C).In data above explanation disodium hydrogen phosphate lipid impaired physical performance, exogenous TAMs grows to oophoroma Impact.TAMs also shortens the time-to-live of tumor-bearing mice simultaneously, but disodium clodronate liposome can be by time lengthening (figure 3D).Additionally, TAMs substantially increases CD68 in the quantity of spheroid and volume, spheroid⁺Macrophage and Ki67⁺Tumour cell but phosphoric acid Sodium salt can weaken above-mentioned effect (Fig. 3, E-G).TAMs equally increases adhesion or is centered around CD68 in spheroid⁺Around macrophage Ki67+ tumour cell quantity.But, clodronate treatment can reduce Ki67⁺Cell and CD68⁺Macrophage quantity (Fig. 3, H-J).

These results suggest that, macrophage is formed for ovarian cancer interior spheroid and cell proliferation is required.

2.4 In TAMs, EGF is mutually raised with EGFR expression in tumour cell and oophoroma growth is played a key effect

Promote the molecular mechanism of OC propagation in order to study TAMs, we pass through transwell measuring F4/80⁺CD206⁺ , to the action effect being isolatable from spheroid ID8 cell (PE-ID8) in abdominal cavity, in experimentation, macrophage and tumour are thin for M2TAMs Born of the same parents do not have directly contact.The external ID8OC cell (primary ID 8) having cultivated is as a control group.As shown in Figure 4 A although F4/ 80⁺CD206⁺TAMs only has faint effect to primary ID 8 cell, finds F4/80 after calculating TCS⁺CD206⁺TAMs is obvious Increase PE-ID8 cell growth.Find F4/80 using Ki67 immunostaining⁺CD206⁺TAMs promotes tumor cell proliferation (Fig. 4, B- C).

It is concluded that TAMs provides growth factor EGF to promote spheroid to be formed and tumor cell proliferation in spheroid.Thus we Using quantitative RT-PCR method to the multiple growth factor expression sides in TAMs and ID8 (PE-ID8) cancer cell extracting from abdominal cavity Formula is screened.Find that only EGF can be detected in TAMs, and other growth factors such as FGFs, HGF, IGF, TNF-α, TGF-β or VEGFs but can not (Fig. 4 D).Detect the F4/80 finding EGF in spheroid source property by quantitative RT-PCR⁺CD206⁺TAMs And great expression in the PE-ID8 of aspheres source property.On the contrary, EGFR high expression in the property PE-ID8 cell of spheroid source, rather than TAMs Interior (Fig. 4 E).Spheroid immunostaining is found with TAMs, energy specific detection is to by EGFR⁺EGF (the figure that tumour cell surrounds 4F).Really, ELISA method detection finds that in ascites, secreting type EGF is maintained at higher concentration (Fig. 4 G) compared with EGF in blood plasma.With The result that qRT-PCR draws is consistent, is not detected by EGF in the supernatant of culture spheroid source property PE-ID8.EGF is in F4/80⁺ CD206⁺Detect in the supernatant of TAMs, and quantity substantially increases (Fig. 4 H) after co-culturing with ID8 tumour cell.

Next we are to F4/80⁺CD206⁺Whether TAMs promotes PE-ID8 tumor cell proliferation to enter by EGF EGFR axle Row measures.For this reason, being degraded expression in TAMs for the EGF with siRNAs, EGFR signal transduction pathway in contrary PE-ID8 is used EGFR antagonist Tarceva blocks.Make EGF silenced gene expression in TAMs by two sets of siRNAs and with qRT-PCR and (Fig. 4, I-J) is examined in the inspection of ELASA method.Verify that Tarceva is intracellular to ID8 by the phosphorylation of EGFR and ERK1/2 Effect (Fig. 4, the K-L of EGF EGFR signal path.It is important that, so that the EGF silence in TAMs is expressed or use Lip river in distress ID8 is carried out treat the PE-ID8 tumor cell proliferation (Fig. 4, M-N) that can be passivated TAMs stimulation completely for Buddhist nun.

2.5 Clinical correlation between OC patient EGF+TAMs and EGFR+ tumour cell and spheroid formation

Formed and EGF to inquire into spheroid observed by us⁺The EGFR that TAMs and OC exists in the patient⁺Tumour is thin Born of the same parents are relevant, and we are detected to 128 spheroids extracting from OC patient's ascites.CD68 is carried out with IHC dyeing display huge Phagocyte occurs nearly in all spheroids of our collections.Additionally, the quantity of macrophage is significantly more than carcinoma in situ in spheroid The quantity (Fig. 5, A-B) of middle macrophage.Similar to mouse model, most of CD68⁺Macrophage collection is from ball centre (figure 5C), illustrate that macrophage plays an important role in spheroid initial formation process in OC Implantation matastasis.To from ascites different size Separate, in (little, neutralization is big) spheroid, CD68 and Ki67 obtaining to be dyeed, they are the mark of macrophage and proliferative cell Thing, finds to be in obvious positive correlation (Fig. 5, C-D) between TAMs and hyperplasia tumour cell.

For understanding contact that may be present between TAM correlation spheroid and clinical pathology in oophoroma, we are high to 128, in, In OC patient's spheroid of low tissue differentiation, TAMs percentage is analyzed.CD68 in OC patient's spheroid⁺Cell quantity with Lymph vessels invade (LVI；P=0.013), ascites volume (p=0.009) and S-CEA 125 (CA-125:A kind of Female ovarian cancer early sign thing and disease occurrence risk have high correlation) (p=0.0043) being proportionate property.To high, in, CD68 positive cell in low differentiation OC patient's spheroid carries out quantization and can be seen that：In differentiation spheroid, its CD68 is positive thin from high to low Born of the same parents' percentage substantially increases, and illustrates that low differentiation OC can attract more macrophages in spheroid is formed compared with differentiated OC (Fig. 5, E-F).Survival analysis display spheroid in CD68 positive cell account for higher percent (>14.5%) patient its 5 years is overall Survival rate (OS) significantly lower than CD68 positive cell account for lower percentage (<14.5%) patient (Fig. 5 G).

2.6 block EGFR in mouse model reduces spheroid formation and oophoroma growth

Formed and EGF in view of spheroid⁺TAMs and EGFR⁺The interaction of tumour cell is relevant, and this is in mouse model and people All such in OC patient, can we block to EGFR retarding agent, and spheroid is formed and tumour growth detects.Tarceva (EGFR retarding agent) suppresses the growth of most of ovarian cancer cell lines, opposite to that, Gefitinib and Cetuximab then suppressing portion Divide ovarian cancer cell line.We select Tarceva that EGFR role during oophoroma Implantation matastasis to be described.For This, we demonstrate, in above-mentioned ID8 model and xenograft mouse model, the operative condition that Tarceva is in progress to OC first, Heterograft be SKOV3 people OCs through gained in Intraperitoneal injection female receptor mouse model, Tarceva is noted together with tumour cell Enter in Recipient mice abdominal cavity.Similar Syngenic mice model, removes the growth of TAMs descendant OC with disodium clodronate liposome (LC) Speed substantially slows down.Additionally, Comparatively speaking giving LC treatment (after tumour cell is implanted 2 weeks) in early days with late period (4-8 week) injection LC When its inhibitory action more notable (Fig. 6 A) to tumour growth.Similar with LC, early stage injection EGFR retarding agent Tarceva pair Thoroughly the growth of passivation oophoroma has more preferable effect (Fig. 6 B).Tarceva treatment group and non-treatment group phase in two groups of mouse models Significantly reduce the raising (Fig. 6, C-F) of simultaneous survival rate than, the former ascites volume and tumor weight.

We analyze spheroid formation, cancer cell multiplication and the ovarian cancer progress situation of Tarceva treatment group further. Accept its number of spheres of mouse group of Tarceva treatment and volume all significantly reduces (Fig. 6, G-I).Tarceva can significantly subtract Dissociate less or TAMs and Ki67 in spheroid⁺The sum (Fig. 6, J-K) of proliferous type cancer cell.These results illustrate TAM secretion EGF shifts the aspects such as the formation of spheroid, cancer cell multiplication and tumour growth in oophoroma early implantation and plays an important role.

2.7EGF promotes EGFR+ tumor cell migration and tumour-TAM spherical by VEGF-C/VEGFR3 signal pathway Become

Next how the EGF that we produce to TAMs mediates spheroid formation is tested, and EGF to the growth of initial OC is Requisite.Data display TAMs is located at cell mass central authorities, and in OC model, EGFR inhibitor Tarceva can significantly reduce ball The quantity of body and volume.Based on above-mentioned discovery, we make TAMs secreting type EGF mediate tumor cell to TAMs migration and sticks It is assumed that this is one of prerequisite that spheroid is formed.Promote the molecular mechanism of OC migration in order to make TAMs clear, we are respectively Detect F4/80 with scarification and transwell method⁺CD206⁺TAMs to spheroid in abdominal cavity (PE-ID8), make by detached ID8 cell With.F4/80⁺CD206⁺TAMs, and non-monocytic cells, are all obviously promoted PE-ID8 cell migration (Fig. 7, A- in two groups of experiments D).But, after in TAMs, the expression of EGF gene silencing or ID8 accept Tarceva treatment, the tumor migration under TAMs stimulates is imitated Should be by dull (Fig. 7, E-F).These results show that in TAMs, EGF and EGFR expression in tumour cell are mutually raised in TAMs rush Play a crucial role in the ovarian tumor cell migration making.

It has been reported that VEGF-C/VEGFR3 signal transduction pathway promotes tumor cell migration in lung cancer model.We send out In ID8 cell, VEGFR3 is in higher level now.It is interesting that we detect in the ID8 (PE-ID8) of abdominal cavity source property VEGF-C, and do not have this to find (Fig. 8, A-B) in primary ID 8 cell or TAMs.Immunostaining results show further VEGF-C and VEGFR3 height in ID8 tumour cell is expressed and is not had (Fig. 9 A) in spheroid TAMs.Immunoblotting assay method PE-ID8 is compared with non-activated ID8 cell for display, and the former intracellular VEGF-C and phosphorylation VEGFR3 level increase (Fig. 8, C- D).It is presumed that EGF induction VEGF-C expression, it activates the VEGFR3 in oncocyte in turn again.Really, 8 are treated through EGF After week or longer time, find mouse ID8 and people through after qRT-PCR (accompanying drawing 8, E-F) and Western blotting (Fig. 8, G-H) detection VEGF-C level in SKOV3OC cell substantially increases；This induction can be blocked by EGFR retarding agent or ERK/2 retarding agent (Fig. 8, I).As before, VEGFR3 phosphorylation can be made through EGF or VEGF-C treatment, this activation is by EGFR antagonism Agent Tarceva or VEGFR3 antagonist MAZ51 are blocked.But, the VEGFR3 activation being induced by VEGF-C but can not be by EGFR Blockers block (Fig. 8, J), illustrates that EGF/EGFR signal transduction pathway plays in VEGF-C/VEGFR3 signal path upstream and makees With.Functionally, EGF and VEGF-C all can promote ID8 cell to shift, and this is consistent with signal transduction result, VEGFR3 antagonist Block the ID8 cell migration (Fig. 9, B-C) of EGF and VEGF-C- induction.These results illustrate EGF EGFR VEGF-C VEGFR3 Signal bypass plays a key effect in adjustment OC cell migration.

Finally, we determined that how TAMs promotes spheroid to be formed.For this reason, we pass through standard 3D co-culture system setting up Play an individual outer sphere test, people CD14+TAMs and from suffering from cancer donor tomato^lyz-creThe GFP isolating in mouse spheroid⁺F4/ 80⁺CD206⁺TAMs mixes (TAM with ID8 cell in the culture medium containing 2% matrigel:ID8 ratio is 1:10) then inoculate To on 24 well culture plates being covered with matrigel.In this model, after co-culturing within 48 hours, we detect spheroid and are formed.For true Determine tumor cell migration to be formed the need of spheroid, we are in 3D co-culture system to EGF siRNA and spheroid in TAMs EGFR or VEGFR3 Idiotype antagonist effects are checked.In mouse cell 3D co-culture system, EGF gene is in TAMs Middle silence expression and blocking-up EGFR or VEGF-C/VEGFR3 signal path can be greatly reduced spheroid volume and quantity (figure 9,D-F).Importantly, the TAMs isolating in people OC patient and SKOV3 cell is tested under 3D co-culture system (Fig. 9, G-I), has drawn similar results.We carry out EGF siRNA in people and mouse cell, detect be dispersed in distribution active TAMs and small sized tumor cell mass (Fig. 9, D and G), illustrate between TAMs and tumour cell contact by suppress EGF/EGFR and VEGF-C/VEGFR3 signal path and be blocked.

2.8 EGF promotes EGFR+ tumour cell mutually glutinous with TAMs by the interaction of ICAM-1- α M beta 1 integrin Attached

It is presumed that it is the necessary premise that orbicule is formed that tumour cell and TAM mutually stick.It is reported that orbicule In TAMs provide integral protein promote itself sticking and between tumour cell.Therefore we pass through fluorescent quantitative PCR technique to from Multiple integral protein expression ways in the ID8 ovarian cancer cell (PE-ID8) isolated in abdominal cavity and TAMs are screened. As expected it has been found that integral protein α M (also referred to as CD11b) and β 2 is in the related F4/80 of orbicule⁺CD206⁺High in TAMs Expression, rather than in tumour cell (Figure 10 A).Facs analysis display CD11b and CD11c is equally in TAM high expression (Figure 10 B- C).It is known that macrophage is being attached to by Mac-1 (being formed by integral protein α M β 2) during inflammatory tissue On Tissue-cell culture (ICAM-1) on vascular endothelial cell or ICAM-2.We detect ICAM-1 and ICAM-2 spherical It is found that ICAM-1 after internal expression, but is not ICAM-2, after detecting by qRT-PCR, find ICAM-1 in spherical internal height Expression (Figure 11 A).Next we are determined by immunostaining technique is which type of cell in spherical internal expression actually ICAM-1.Make us surprisingly, ICAM-1 high expression (Figure 12 A) not in TAMs but in ID8 tumour cell. .ICAM-1 the baseline expression level in primary ID 8 cell is low-down, but either mRNA or egg under EGF effect White matter level all significantly raises (Figure 11 B-D).EGFR- or ERK1/2 retarding agent can stop the ICAM-1 expression (figure of EGF induction 12, B-C).It is interesting that the ICAM-1 expression of EGF induction also can be blocked by VEGFR3 retarding agent.VEGF-C induction (Figure 12, D-E) that expression in ID8 cell for the ICAM-1 is consistent with.

Hereafter we to the ICAM-1 interphase interaction on the integral protein α M β 2 and tumour cell on TAMs whether to first Beginning spheroid forms to play a crucial role and is detected.For this reason, we determine ICAM-1 neutralizing antibody forms experiment in 3D spheroid In effect, ICAM-1 neutralizing antibody substantially reduces mouse (Figure 12, F-H) and people's cell (Figure 12, I-K) in 3D co-culture system The quantity of middle spheroid and volume, but the IgGs as reference does not have this phenomenon.It should be noted that anti-ICAM-1 antibody pair The function influence of spheroid is more deep compared with making the expression of EGF gene silencing or blocking-up EGFR/VEGFR3 conduction path, and TAM is described It is the step that spheroid forms middle early stage that tumour cell is combined.In order to seek effect in treatment of ovarian cancer for the ICAM-1 neutralizing antibody Really, in the orthotopic mouse model of foundation, ID8 tumor-bearing mice gives anti-ICAM-1 antibody or the IgGs as comparison to be controlled Treat.It is swollen that phenotype analytical shows that anti-ICAM-1 antibodyome significantly reduces that Mouse Weight, ascites volume and separating from ascites obtain Oncocyte weight in wet base (Figure 12, L-N).Similar with EGFR retarding agent effect, anti-ICAM-1 antibody significantly reduces quantity and the body of spheroid Long-pending (Figure 12, O-Q).These results illustrate that the TAM- tumour of integral protein-ICAM-1 mediation is combined and are formed and oophoroma in spheroid Play an important role during Implantation matastasis.

3. Analysis of conclusion

Almost there is with all OC patients (more than 90%) in plantation (abdominal cavity) transfer, be especially common in TCA and suffer from Person simultaneously thus leads to death.Implantation matastasis also betide other many cancers such as cancer of pancreas (50%) and colorectal cancer (32%).Cause This, the Common Mechanism of research Implantation matastasis is most important to the prognosis improving OC and other Implantation matastasis cancer patient.Consider Peritonaeum has the characteristics that its unique such as space is larger, no blood vessel or lymphatic.How the OC cell that comes off obtains necessary matrix Support and avoid anoikis generation, its avoid the appropriate protection mechanism of immune cells attack and which specificity give birth to The long factor can maintain them to mushroom out and implantation do not study yet at present clear.Once had been reported that the survival of OC cell energy active cell was logical Surface immunosuppression molecule is raised (as B7-H4 and complement as activated RAB25 thus obtaining anchorage dependence survival ability in road simultaneously C1 mortifier) carry out the attack (as T cell or complement) of protected from immune system.It is that OC peritoneal implantation metastasis are opened that orbicule is formed Dynamic another basic step.But, the detailed mechanism being formed with regard to orbicule is still not clear.By to 128 III phase OC patients In ascites, the cell component of detached composition orbicule is analyzed it has been found that macrophage is present in all orbicules. We have also observed that contained macrophage numbers in detached orbicule from ascites are significantly more than in primary tumo(u)r huge bite thin Born of the same parents' quantity.In addition it has been found that number of macrophages amount is proportionate in spherical internal hyperplasia with it, but the prognosis with OC patient In negative correlation.Our result show that perhaps orbicule correlation TAMs plays an important role in mankind OC progress.

TAMs can promote Tumor angiogenesis by secreting the angiogenesis promoting factor and chemical inducer in solid tumor And cancer metastasis, TAMs also can secrete cytokines promote metastases before microenvironment formation.Macrophage institute in OC Work and carried out research in the mouse model set up before this.Oophoroma peritonaeum during inflammation diffusion in the report such as Toni abdominal cavity The danger of transfer will increase；Consume peritoneal macrophages, but do not include neutral grain and NK, peritonaeum can be reduced The probability that Implantation matastasis occur.These results and the result that we observe from human sample all point out us to make a kind of vacation If：Macrophage plays vital effect during spherical body is formed before peritonaeum transfer occurs.The OC sample of people and mouse In this, TAMs is respectively positioned on orbicule central authorities (Figure 13 A：TAM- cancer cell is in spherical internal interaction model one), this is interesting Find to support our hypothesis energetically.We are further provided with strong evidence evidence and show that TAMs is formed in initial step in orbicule Essential.In the OC carcinoma in situ model set up using GFP transgene receptor mouse, it is observed that penetrate into the huge of abdominal cavity biting Cell there are about 80% and occurs in spheroid.After TAMs is using clodronate therapy, the size and number of sphere cells significantly subtract Few, in turn, abdominal cavity field planting is suppressed prolonged survival period.On the contrary, spheroid after isolated TAMs injects in mouse peritoneal Volume is significantly increased with quantity.It is interesting that the macrophage in infiltration abdominal cavity gradually obtains M2 sample phenotype.

In OC mouse model, it is observed that Implantation matastasis occur after 3 weeks inner tumour cell numbers growth with swell Knurl orbicule forms relevant.Really, observe that the related spherical physical efficiency of TAM promotes the increasing of tumour cell in mouse and people's OC sample Raw.In addition, finding that TAMs can directly facilitate tumor cell proliferation in external transwell test, show that TAMs passes through to discharge certain A little regulatory factors carry out the growth of modulate tumor.By screen one group of growth factor in TAMs and spheroid associated tumor cells and it Homoreceptor, we have confirmed that EGF/EGFR regulatory pathway stimulates lower OC hyperplasia to TAM and tumour progression has to closing and weighs The effect wanted.Early-stage Study has been reported expression in EGFR Iisolated tumor cells in abdominal cavity and has been determined to compare in tumour cell substantially Increase, illustrate that EGFR path plays an important role in abdominal cavity Iisolated tumor cells.Week, abdomen in ovarian cancer patients abdominal cavity known to crowd There are a large amount of macrophages and T lymphocyte in water.Additionally, it has been reported that breast cancer cell can be promoted from the EGF of macrophage Invasion.Our research clearly illustrates that TAM is the main source of EGF, the positive OC propagation of its induction EGFR, and this exists Confirmed in mouse model in vitro and by EGF gene silencing and EGFR retarding agent.Under abdominal cavity microenvironment, EGF is in TAM Waiting of how being adjusted is studied further.Interaction in spheroid for the TAM-OC cell probably increased the table of EGF Reach.Similarly, the spheroid correlation intracellular EGFR level of OC separated from ascites significantly raises.

It is interesting that EGFR- positive OC cell is looped around around EGF- positive TAMs in spheroid.This close contact is said The TAMs of bright ball centre is not only OC cell and avoids anoikis to provide initial matrix support, by EGF-EGFR Secretion loop promotes the propagation of OC cell.Additionally, we demonstrate that EGF/EGFR axle not only plays an important role to OC propagation, also lure Lead VEGF-C in the intracellular expression of OC, it activates the bypass of VEGFR3 autocrine in turn and then improves integral protein/ICAM-1 Expression, the migration of OC cell and spheroid form (Figure 13 B).This conclusion is drawn by following research：1) in transwell Find when being co-cultured with TAMs cell by reducing EGF value or to significantly inhibit OC using EGFR retarding agent Tarceva thin The propagation of born of the same parents.Additionally, observing that in two OC mouse models EGFR retarding agent can thoroughly be passivated orbicule formation, OC cell increases Grow and tumour growth；2) EGF that TAM produces induces the expression of the intracellular VEGF-C of OC, and it activates VEGFR3 bypass in turn；Logical Cross retardance EGFR-ERK signal transduction can eliminate OC intracellular by EGF induce produce VEGF-C.Importantly, EGF and VEGF-C Expression in OC cell for the ICAM-1 and the differentiation of OC cell all can be stimulated；Above-mentioned all effects are using VEGFR3 retarding agent Afterwards all can be weakened.Therefore, act on the downstream portion of EGFR signal transduction (see figure in the intracellular VEGF-C/VEGFR3 of OC 13B).

Our data showed that early stage consume TAMs or with Tarceva block OC cell in EGFR can be effectively pre- The implantation metastasis of anti-oophoroma, but late above-mentioned measure is invalid.Perhaps, this result of study can explain why late period OC suffers from Person fails to bear fruit using EGF antibody single therapy.Therefore, the treatment of OC should include early diagnosis, removal TAMs and EGF resists The use of autogenic therapy.Additionally, EGF/EGFR, VEGF-C/VEGFR3 and ICAM-1 α M β 2 integral protein model that we set up will Ask OC cell proliferation, migration, adhesion and spheroid to be formed and there is clinical meaning.Therefore, ICAM-1 neutralizing antibody, similar to blocking-up EGF and VEGFR3 signal transduction, the formation of the external spheroid of strong inhibition.And, ICAM-1 neutralizing antibody significantly reduces mouse mould The progress of the size of spheroid, quantity and tumour in type.The study show that neutralization ICAM-1 may provide new treatment for treatment OC Method.

Generally speaking, our research indicate that TAMs plays in oophoroma Implantation matastasis orbicule forming process must Few effect.TAM can secrete a large amount of EGF thus activating the EGFR in surrounding tumor cells.The EGF/EGFR signal path of activation VEGF-C can be raised, and it raises integral protein and ICAM-1 in turn thus forming autocrine regenerative feedback loop again, thus promoting swollen Oncocyte hyperplasia, differentiation, adhesion, formation orbicule and peritonaeum transfer (see Figure 13 B).We disclose orbicule at current research Formed key mechanism, for suppression Implantation matastasis provide a kind of New Policy and the prognosis situation improving OC patient.

Last should be noted that above example is only in order to illustrate technical scheme rather than to present invention guarantor The restriction of shield scope, although being explained in detail to the present invention with reference to preferred embodiment, those of ordinary skill in the art should Understand, technical scheme can be modified or equivalent, without deviating from the essence of technical solution of the present invention.

Claims (10)

1.VEGF-C screening for diagnose or treat Implantation matastasis cancer medicine in purposes.

2. purposes as claimed in claim 1 is it is characterised in that the Implantation matastasis of described Implantation matastasis cancer and macrophage phase Close.

3. purposes as claimed in claim 1 is it is characterised in that described Implantation matastasis cancer is oophoroma, cancer of pancreas or colorectal cancer.

4. purposes as claimed in claim 1 is it is characterised in that described medicine passes through to reduce VEGF-C level, retardance VEGF-C Acceptor or blocking VEGF-C downstream signal treating Implantation matastasis cancer.

5. purposes as claimed in claim 4 is it is characterised in that the downstream signal of described blocking VEGF-C passes through to suppress ICAM-1 Come to carry out.

6. purposes as claimed in claim 1 is it is characterised in that described medicine passes through to reduce VEGF-C level or retardance VEGF-C Acceptor and suppress the formation of the migration of EGFR positive tumor cell and tumour cell-TAMs spheroid treating Implantation matastasis cancer.

The downstream signal antagonistic of 7.VEGF-C inhibitor, the beta blocker of VEGF-C or VEGF-C is in preparation for treatment kind Plant the purposes in the medicine of metastatic carcinoma.

8. purposes as claimed in claim 7 is it is characterised in that described VEGF-C inhibitor is VEGF-C neutralizing antibody.

9. purposes as claimed in claim 5 is it is characterised in that the beta blocker of described VEGF-C is VEGFR3 antagonist MAZ51.

10. purposes as claimed in claim 5 is it is characterised in that the downstream signal antagonistic of described VEGF-C is in ICAM-1 And antibody.