Animal models for amplifying human or animal circulating tumor cells

Abstract

The present invention relates to an avian model that allows the expansion of human or animal circulating tumor cells (CTCs) and its use for monitoring and determining the sensitivity of patients or animals suffering from cancer to one or more therapeutic agents, and for screening new therapeutic agents intended for the treatment of cancer.

Description

[Technical field]

[0001] The present invention relates to an avian model that allows the expansion of human or animal circulating tumor cells (CTCs). [Background technology]

[0002] CTCs are cells that can detach from tumors and enter the vascular system before being displaced to enter the blood. These cells can therefore migrate to other organs and participate in the development of metastasis. Thus, these cells represent a very attractive target in monitoring the evolution and treatment of cancer, or even in the discovery of novel therapeutic approaches.

[0003] However, the isolation of CTCs is not a simple task, since the numbers of these cells are very low. Therefore, it is necessary to expand them before studying them. Currently, CTCs can be expanded using in vitro or in vivo culture in mouse models (Kowalik A et al., 2017; Drapkin BJ et al., 2018). In vitro culture of CTCs derived from numerous cancers (lung, prostate, breast, urinary tract, head or neck cancer, etc.) has already been performed, making it possible to perform fast preclinical testing of treatment plans, but these cultures are too long to be compatible with clinical practice and expose CTCs to phenotypic modifications, so the success has been limited (Pantel K, Alix-Panabieres C., 2016).

[0004] Moreover, short-term in vivo cultures were established in mouse models from CTCs and after a first step of in vitro expansion when CTC amounts were too low (Giuliano M et al., 2015; Williams ES et al., 2015; Torphy RJ et al., 2014; Rossi E et al., 2013). Direct inoculation of CTCs into immunodeficient mice has also been envisaged as a method for amplifying isolated CTCs. In highly metastatic small cell lung cancer (SCLC), CTCs isolated in patients with high levels of CTCs (more than 400 CTCs per 7.5 mL of blood) formed tumors in mice, and the response of these tumors to platinum- and etoposide-based chemotherapy was consistent with clinical observations (Hodgkinson, CL et al., 2014). These studies pave the way for the use of immunodeficient mice as "incubators" of CTCs, capable of generating preclinical models suited to the individual tumors of the patient.

[0005] However, these models are too expensive to envisage their systematic use. They often require the implementation of a prior step of in vitro expansion, which has the aforementioned drawbacks. Finally, the establishment of these models requires time, which is not always compatible with the observation of the desired effect. Summary of the Invention

[0006] There is therefore a need to find a cheaper in vivo model, but one that also gives results more quickly and does not require a prior in vitro expansion step, in order to address these CTCs closest to those present in vivo in patients.

[0007] The present invention relates to an animal model for studying circulating tumor cells, consisting of an avian embryo-containing egg, preferentially a chicken, comprising human or animal circulating tumor cells (CTCs) isolated from a sample of a patient or animal suffering from cancer, said CTCs being implanted at the level of the chorioallantoic membrane (CAM) of the embryo-containing egg, said embryo-containing egg being at a developmental stage corresponding to the formation of the CAM and corresponding to at least 8 days of chicken development at the time of implantation.

[0008] This embryo-containing egg is an animal model, more specifically an avian model, for amplifying CTCs.It also allows to study the effect of various drugs on tumorigenesis to select those that have anticancer activity and thus may represent effective therapeutics for treating the patient or animal that the CTCs implanted in the embryo-containing egg are derived from.This allows to administer to the patient therapeutic agents that have been pre-tested against the cells of the patient's own tumor, thus leading to personalized treatment.

[0009] The present invention also relates to a method for the preparation of this avian embryo-containing egg model allowing the expansion of human or animal circulating tumor cells (CTCs) obtained from a sample of a patient or animal suffering from cancer, said method comprising transplanting CTCs isolated from said sample at the level of the chorioallantoic membrane (CAM) of an embryo-containing egg, said embryo-containing egg being at a developmental stage corresponding to the formation of the CAM and corresponding to at least 8 days of chicken development.

[0010] The present invention also relates to a method for expanding human or animal circulating tumor cells (CTCs) isolated from a sample of a patient or animal suffering from cancer, comprising the steps of: a) preparing an embryo-containing egg model according to the invention by transplanting CTCs isolated from a sample of a patient or animal suffering from cancer at the level of the chorioallantoic membrane (CAM) of an avian embryo-containing egg, said embryo-containing egg being at a developmental stage corresponding to the formation of the CAM and corresponding to at least 8 days of chicken development; b) harvesting tumors arising from the expanded CTCs, and optionally, c) recovering CTCs from the tumor collected in step b). The method comprises the steps of: Preferably, multiple transplants, advantageously two transplants, may be performed within the scope of this method to further optimize the expansion of CTCs.The method thus comprises performing a secondary transplant of the tumor derived from the primary transplant of CTCs into the CAM of a first embryo-containing egg into the CAM of a second embryo-containing egg.

[0011] The present invention also provides a method for determining the susceptibility of a patient or animal suffering from cancer to one or more therapeutic agents, comprising the steps of: amplifying circulating tumor cells (CTCs) obtained from a sample of a patient or animal suffering from cancer according to the invention by transplanting CTCs isolated from said sample at the level of the chorioallantoic membrane (CAM) of an embryo-containing egg, said embryo-containing egg being at a developmental stage corresponding to the formation of the CAM and corresponding to at least 8 days of chicken development, Implanting an embryo-developed tumor at the level of the chorioallantoic membrane (CAM) of a new avian embryo-containing egg, said new embryo-containing egg having been previously incubated to a developmental stage corresponding to the formation of a CAM and corresponding to at least 8 days of chicken development; administering said therapeutic agent to said embryo-containing egg at least 12 hours after said transfer; and studying the effect of the therapeutic agent administered thereby on the tumorigenesis of tumors arising in the newly implanted embryo-containing eggs. The present invention also relates to a method comprising the steps of:

[0012] Preferably, this method for determining the sensitivity of a patient or animal suffering from cancer to one or more therapeutic agents further comprises the steps of incubating the implanted embryo-containing egg for at least one hour after administration of said agent, and optionally, harvesting the tumors that develop from the implanted CTCs at the end of incubation of said new implanted embryo-containing egg.

[0013] The present invention further provides a method for monitoring a subject, a patient or animal, suffering from cancer, comprising the steps of: preparing a first avian embryo-containing egg as described above using the CTCs isolated from said patient or animal sample at T1 and studying the tumorigenesis of tumors arising in said first embryo-containing egg; preparing a second avian embryo-containing egg, as described above, using CTCs isolated from the same patient or animal sample at time T2, and studying the tumorigenesis of tumors developing in this second embryo-containing egg; Comparing the tumorigenesis of tumors developed in said first embryo-containing egg and said second embryo-containing egg. The method comprises the steps of:

[0014] Finally, the present invention relates to a method for screening a therapeutic agent for in vivo treatment of cancer, comprising: preparing an embryo-containing egg model by implanting CTCs isolated by the above method from a sample of a patient or animal suffering from cancer at the level of the chorioallantoic membrane (CAM) of an embryo-containing egg, said embryo-containing egg being at a developmental stage corresponding to the formation of the CAM and corresponding to at least 8 days of chicken development; administering one or more candidate agents to said embryo-containing egg at least 12 hours after said transfer; and studying the effect of the administered therapeutic agent on tumorigenesis of tumors arising in the implanted embryo-containing eggs. The method comprises the steps of: [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 depicts a diagram of the embryo-containing egg model with the CTC implantation site in the upper CAM. [Diagram 2] Figure 2 depicts a possible experimental setup for implementing the present invention: First transplant (first egg set): CTCs isolated from samples collected from patients are transplanted in one egg per patient. Second transplant (second egg set): A secondary transplant allows a new amplification of the tumor collected in the egg after transplantation. [Diagram 3] Figure 3 shows in ovo images of tumor growth obtained by xenografting on CAM during the first round of amplification of CTCs isolated from the blood of patients suffering from lung, breast or prostate cancer. [Figure 4] FIG. 4 represents a histological section of a tumor derived from CTCs isolated from the blood of a patient suffering from breast cancer that was implanted onto the CAM during the first round of amplification. [Diagram 5] FIG. 5 shows the results of genetic analysis of the human tumor markers TP53 (A) and KRAS (B) on tumors derived from CTCs of patients suffering from lung cancer (#SH103 and #CM105) implanted on the CAM at the time of the first amplification. [Figure 6] Figure 6 represents in ovo images of tumor growth derived from CTCs isolated from the blood of a patient suffering from lung cancer, implanted onto the CAM at the second amplification (secondary implantation). [Figure 7] Figure 7 shows in ovo images of tumors obtained from CTCs isolated from the blood of a patient suffering from prostate cancer after primary (A) and secondary (B) transplantation, as well as histological analysis of tumor sections showing the amplification of in ovo transplanted CTCs after secondary transplantation (C). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention relates to an animal model, in particular an avian model, for studying circulating tumor cells, consisting of an avian embryo-containing egg comprising human or animal circulating tumor cells (CTCs) isolated from a sample of a patient or animal suffering from cancer, said CTCs being implanted at the level of the chorioallantoic membrane (CAM) of the embryo-containing egg, said embryo-containing egg being at a developmental stage corresponding to the formation of the CAM and corresponding to at least 8 days of chicken development at the time of implantation.

[0017] Preferably, the embryo-containing egg according to the present invention is from the order Galliformes ( Galliformes ) or Struthioniformes ( Struthioniformes) bird eggs. In particular, the eggs are particularly preferably those of birds of the Galliformes order, in particular chickens, quails, turkeys, pheasants, peacocks, guinea fowl or other farm birds. They may also be ostrich eggs. Advantageously, the embryo-containing eggs according to the invention are chicken ( Gallus gallus ) eggs.

[0018] Within the scope of the present invention, the term "embryo-containing egg" denotes a fertilized avian egg in which the embryo can develop under suitable conditions, in particular in an incubator at a temperature of 37° C. to 38° C. Under these conditions, the incubation time until the egg hatches is 21 days in chickens.

[0019] The developmental stages taught herein are defined as a function of incubation time after fertilization of the egg, in particular incubation time under suitable conditions as defined above. "Implantation at the level of the CAM" is used to indicate administration by apposition or injection into the CAM, whether this is the upper or lower CAM.

[0020] The embryo-containing egg model according to the present invention has cells derived from two different organisms or xenografts: "host" or "recipient" avian cells and "recipient" CTCs implanted into eggs derived from a human or animal organism of a different species than the avian. In a particularly preferred mode, the CTCs implanted into the avian embryo-containing egg are human cells. These implanted CTCs then develop in the embryo forming one or more solid tumors and / or replacing them within the egg.

[0021] Obviously, the "implantation at the level of the CAM" is carried out after the formation of the CAM, at a stage corresponding to at least 8 days of development in chickens under normal and standard growth conditions. If the bird used is a chicken, this stage corresponds to at least 8 days of development. The number of days of development may vary from species to species, and the transplantation is carried out after changing the number of days of development. For example, a developmental stage of at least 8 days in chickens corresponds to a developmental stage of at least 6.5 days in quails.

[0022] "A sample of a patient or animal suffering from cancer" should be understood to mean any sample containing CTCs derived from a human or animal suffering from cancer. Preferably, said sample is selected from whole blood or biological fluids that may contain CTCs, such as pleural fluid, ascites and cerebrospinal fluid (CSF), preferably whole blood. Thus, the CTCs to be implanted in the embryo-containing egg may be derived from any type of cancer that produces CTCs in blood or this type of biological fluid, in particular from metastatic cancer. According to a preferred embodiment of the present invention, the patient or animal from which the CTCs to be implanted in the embryo-containing egg are isolated suffers from at least one cancer selected from lung cancer, prostate cancer, breast cancer, colorectal cancer.

[0023] Advantageously, the embryonated egg model according to the invention is a chicken egg in which human CTCs are implanted at the level of the CAM after at least 8 days of development.

[0024] It is understood that the embryos implanted according to the present invention have no mission to hatch and are therefore not intended to produce adult organisms.For this reason, the embryos implanted according to the present invention are models intended to receive CTCs during their amplification period, which does not extend to hatching, corresponding to the 21-day development of chickens.Optionally, the avian embryos according to the present invention are sacrificed before hatching and after the implanted CTCs have caused the development of one or more tumors in the egg, in accordance with the ethical regulations in force.

[0025] Such a model allows the simultaneous expansion of CTCs, in particular by the development of tumors from these CTCs, on which it is possible to test the anti-cancer activity of various agents known to have anti-cancer activity or that are candidates to determine whether they have such activity. The comparative efficacy of multiple therapeutic agents can also be tested to determine the most promising therapy for treating patients who receive transplanted CTCs.

[0026] According to a second aspect, the present invention relates to a method for the preparation of an avian embryo-containing egg model as described above, allowing the expansion of human or animal circulating tumor cells (CTCs) isolated from a sample of a patient or animal suffering from cancer, comprising transplanting the CTCs isolated from said sample at the level of the chorioallantoic membrane (CAM) of an avian embryo-containing egg, said embryo-containing egg being at a developmental stage corresponding to the formation of the CAM and corresponding to at least 8 days of chicken development.

[0027] The skilled artisan will know how to determine when to perform the transplantation of CTCs as a function of the bird species used, i.e. the minimum number of days of incubation or development of the embryo-containing egg to reach the formation of the CAM and a developmental stage corresponding to at least 8 days of development in chickens, for example in chickens the transplantation can be performed from 8 days of development and in quails from 6.5 days of development.

[0028] According to a preferred embodiment, the embryonated egg is incubated prior to implantation to a developmental stage corresponding to the formation of the CAM, which corresponds to at least 9 days of development in chickens, or in an even more preferred manner, at least 9.5 days of development.

[0029] The incubation is carried out under suitable conditions, ie conditions allowing the normal development of the embryo-containing eggs, in particular at a temperature comprised between 37°C and 39°C, preferably at 38°C or even 38.5°C.

[0030] The transplantation of CTCs can be carried out anywhere in the CAM, at the top or bottom, preferably at the level of the top CAM. Any method known to those skilled in the art can be used for this transplantation, in particular the transplantation technique referred to by Crespo P. & Casar B, 2016.

[0031] According to a particular embodiment, the amount of CTCs transplanted will be approximately 5 to approximately 5000 CTCs, in a preferred manner approximately 5 to approximately 2500 CTCs and in an even more preferred manner approximately 5 to approximately 1000 CTCs.

[0032] According to a preferred embodiment, the CTCs used are frozen after isolation from a sample of a patient or animal suffering from cancer and prior to implantation into an embryo-containing egg.

[0033] In the transplantation at the level of CAM, CTCs are isolated from the sample of a patient or animal suffering from cancer by any method known to those skilled in the art.Thus, CTCs are isolated by purifying them from other cells present in the sample from a patient or animal suffering from cancer, in particular by separating them from immune cells present in the sample.

[0034] Methods for isolating CTCs can be based on different principles that allow to separate them from other components of the sample and thus to perform CTC enrichment. A large number of different methods have been described, in particular by Zheyu Shen et al., 2017. They allow to have a so-called "negative" enrichment, when the aim is to capture non-target cells and elute CTCs, or to have a so-called "positive" enrichment, when the aim is to capture CTCs and elute non-target cells of the sample.

[0035] Among these methods, it is possible to cite separation methods by filtration, and in particular by vertical filtration, such as the ISET (Isolation by Size of Tumor Cells) technique, for example sold by the Rarecells SAS Company and described in particular by Han Wei Hou et al., 2013.

[0036] Certain methods may rely on a marking step, such as the CellSearch method (Kagan M, et al., 2002), which is based on the immunoselection of CTCs using ferrofluidic nanoparticles containing antibodies targeting a specific epithelial cell adhesion marker (EpCAM), so that CTCs are magnetically separated from the majority of other blood cells.

[0037] Other methods based on the use of microfluidic systems may also be used, relying on the separation as a function of different parameters such as cell size, shape, density, deformability, etc. Among these, the VTX-1 method as described by Sollier-Christen et al., 2018, the microfluidic method based on the capture of tumor cells expressing the EpCam molecule (CTC-chip) using microspots coated with anti-EpCam antibodies (described in particular by Nagrath S et al., 2007), the RosetteSep technology of the Stemcells Company, which uses a mixture of antibodies (CD45, CD66b and glycophorin A) bound to magnetic beads targeting red and white blood cells, or alternatively the ClearCell Fx1 method developed by the Biolidics Company (formerly ClearBridge), which is described in particular by Laget S et al., 2017 and is particularly advantageous.

[0038] According to a particular embodiment, the method for preparing an avian embryo-containing egg model according to the invention further comprises a step of incubation of the embryo-containing egg once transferred for at least 12 hours, preferably at least 24 hours, and in an even more preferred manner at least 48 hours after transfer, before it is used. Preferably, the embryo-containing egg once transferred is incubated for a maximum of 20 days, in particular up to 18 days, in relation to the developmental stage of the chicken embryo.

[0039] According to a third aspect, the present invention provides a method for the expansion of human or animal circulating tumor cells (CTCs) isolated from a sample of a patient or animal suffering from cancer, comprising the steps of: a) implanting CTCs isolated from a sample of a patient or animal suffering from cancer, as described above, at the level of the chorioallantoic membrane (CAM) of an avian embryo-containing egg, said embryo-containing egg being at a developmental stage corresponding to the formation of the CAM and corresponding to at least 8 days of chicken development; b) harvesting tumors arising from the expanded CTCs, and optionally, c) recovering CTCs from the tumor collected in step b). The present invention relates to a method comprising the steps of:

[0040] The inventors have demonstrated in an unexpected manner that CTCs, in particular CTCs of human origin, can be expanded in an avian embryo-containing egg model, making it possible not only to obtain them in sufficient quantities to study them, but also to use them to guide the identification of novel treatments or to select those likely to be most effective in an individualized manner.

[0041] According to a preferred embodiment of the above-mentioned method for expanding human or animal circulating tumor cells (CTCs), the expanded CTCs can be transplanted again, for example by performing transplantation and collection multiple times (the term secondary transplantation is used for the second transplantation), in order to further improve the expansion of CTCs. Thus, the method includes the following steps: a) implanting CTCs isolated from a sample of a patient or animal suffering from cancer at the level of the chorioallantoic membrane (CAM) of an avian embryo-containing egg, said embryo-containing egg being at a developmental stage corresponding to the formation of the CAM and corresponding to at least 8 days of chicken development; b) harvesting tumors arising from the expanded CTCs; b1) implanting the tumor collected in step b) at the level of the chorioallantoic membrane (CAM) of a second avian embryo-containing egg, said second embryo-containing egg having been previously incubated until a developmental stage corresponding to the formation of the CAM and corresponding to at least 8 days of chicken development, b2) optionally incubating said second embryo-containing egg thus transferred for at least 12 hours; b3) harvesting tumors arising from the tumors implanted in step b1), and optionally c) recovering CTCs from the tumor collected in step b3); may comprise:

[0042] According to a particular embodiment, the above-mentioned method for expanding human or animal circulating tumor cells (CTCs) further comprises a step of incubating the once implanted embryo-containing egg for at least 12 hours, preferably at least 24 hours, and even more preferably at least 48 hours after implantation, before carrying out the collection of tumors derived from the implanted CTCs.Preferably, the once implanted embryo-containing egg is incubated for at most 20 days, in particular up to 18 days, in relation to the developmental stage of chicken embryos.

[0043] Thus, the present invention also provides a method for determining the susceptibility of a patient or animal suffering from cancer to one or more therapeutic agents, comprising the steps of: As described above, the CTCs isolated from a patient or animal sample suffering from cancer are expanded in avian embryo-containing eggs; implanting the tumor thus harvested at the level of the chorioallantoic membrane (CAM) of a fresh avian embryo-containing egg, said fresh embryo-containing egg having been previously incubated to a developmental stage corresponding to the formation of the CAM and corresponding to at least 8 days of chicken development; administering said therapeutic agent to said embryo-containing egg at least 12 hours after said transfer; and studying the effect of the therapeutic agent administered thereby on the tumorigenesis of tumors arising in the newly implanted embryo-containing eggs. The present invention relates to a method comprising the steps of:

[0044] "Therapeutic agent" is taken to denote any chemical or biological molecule or compound, nanostructure, physical method or combination thereof that has an anti-tumor effect, in particular that is potentially effective for treating the type of cancer that has arisen from a tumor-like cell implanted in said embryo-containing egg.

[0045] Such compounds may be chemical molecules such as chemotherapeutic drugs, biological compounds such as antibodies, therapeutic cells such as CART, physical means such as irradiation, intercalating agents such as, for example, irinotecan, tyrosine kinase signal pathway inhibitors such as, for example, sunitinib, anti-hormonal drugs such as, for example, tamoxifen, immunomodulatory agents such as, for example, Ketruda, membrane receptor inhibitors such as, for example, trastuzumab, and the like.

[0046] Within the scope of the present invention, the terms tumor and cancer are used indifferently and interchangeably to define the proliferation of malignant cells. The same applies to the use of the terms anti-tumor and anti-cancer.

[0047] In particular, the fact that the CTCs to be transplanted are isolated from a sample of a patient or animal suffering from cancer makes it possible to test multiple therapeutic agents and select the most promising one for treating the tumor of this patient. Therefore, according to a preferred embodiment of the present invention, the avian embryo-containing eggs transplanted with CTCs are used to determine the agent with the best anti-cancer activity among the various agents tested.

[0048] Avian embryo-containing eggs implanted with CTCs may also be used, according to the present invention, to test the anti-cancer efficacy of combinations of therapeutic agents compared to the effects obtained with each agent tested independently.

[0049] Within the scope of this use of embryo-containing eggs, it is possible to determine or even quantify the toxicity of the tested therapeutic agent both on the tumor developing from the implanted CTCs and on the whole embryo. Therefore, another subject of the present invention relates to the use of avian embryo-containing eggs implanted with CTCs isolated from a sample of a patient or animal suffering from cancer to quantify the toxicity of one or more therapeutic agents on the tumor and / or on the whole embryo.

[0050] The step of administering the therapeutic agent into the embryo-containing egg can be carried out in various ways according to techniques well known to those skilled in the art. Administration can be carried out, in particular, by apposition or injection at the level of the CAM, by intratumoral injection, by injection into embryonic or extraembryonic structures of the embryo-containing egg.

[0051] The administration of the therapeutic agent is carried out at least 12 hours after the transplantation of the CTCs, preferably at least 24 hours after the transplantation, or even more preferably at least 48 hours after the transplantation, i.e. 1-2 days after the transplantation. The therapeutic agent can also be administered according to different treatment regimes, not only in terms of duration but also in terms of the number of administrations, for example, once every two days, or every day, or twice a day, or a single injection, and up to the last day of incubation of the embryo-containing egg. These choices are determined as a function of the agent to be administered.

[0052] According to a preferred embodiment, the method for determining the sensitivity of a patient or animal suffering from cancer to one or more therapeutic agents according to the invention further comprises an incubation of the once implanted embryo-containing egg for at least 1 hour, before studying the effect on tumorigenesis, after administration of the therapeutic agent to the implanted embryo-containing egg. Advantageously, this incubation is carried out for at least 4 days and at most 12 days after administration, so as to correspond to a maximum of 21 days of embryonic development, advantageously 18 days of development. According to a particular embodiment, the method for determining the sensitivity of a patient or animal suffering from cancer to one or more therapeutic agents according to the invention further comprises, at the end of the incubation of said embryo-containing egg after administration of the administered therapeutic agent, collecting the tumors that develop from the implanted CTCs, in particular by microdissection. Thereby, the study of the effect of the administered therapeutic agent on tumorigenesis, in particular after collection of the tumors that develop in the implanted embryo-containing egg, can take several complementary approaches. The methods may in particular comprise the analysis of parameters such as tumor growth, metastatic invasion, angiogenesis, neovascularization, inflammation and / or tumor immune infiltration, toxicity towards the tumor and / or towards the whole embryo.

[0053] Thus, the tumors may be subjected to analyses to measure and / or analyze these different parameters, such as tumor weight and / or volume to study tumor growth, expression of different specific markers to study metastatic invasion (e.g. amplification of Alu sequences by quantitative PCR for human metastases), number of blood vessels within the tumor for angiogenesis and neovascularization, quantification of interleukins for inflammation and / or markers such as CD3, CD8, CD4, CD45 and CD56, in particular by rtQPCR, to assess tumor immune infiltration, weight, and histological analyses to assess toxicity to the tumor.

[0054] Metastatic invasion studies can be performed in the easily accessible lower CAM, but also in any target organ within the embryo, particularly as a function of the type of cancer and known data regarding the associated metastatic phenomenon.

[0055] Inflammation and / or tumor immune infiltration can be studied by analysis of the expression of various markers, such as CD3 (T lymphocyte membrane marker), CD4 (regulatory T lymphocyte, monocyte and macrophage membrane marker), CD8 (cytotoxic T lymphocyte marker), CD45 (leukocyte membrane marker), CD56 (NK cell marker), etc. To avoid interspecies cross-breeding, specific oligonucleotide pairs of these markers can be developed.

[0056] By extension, it may also be possible to monitor inflammation and infiltration of cells of the immune system at the metastatic site.

[0057] The combined analysis of all these factors, well known to those skilled in the art, makes it possible to determine the sensitivity of the patient or animal suffering from cancer from which the transplanted CTCs were obtained, to the therapeutic agent administered to the embryo. These parameters form an integral part of the decision tree used by the clinician to determine the therapeutic management to be adopted in patients suffering from cancer in particular.

[0058] Within all methods according to the invention comprising the effect of a therapeutic agent on tumor formation, these parameters are preferentially evaluated by comparison after administration of the therapeutic agent to an embryo-containing egg once transferred, in comparison with those determined in another embryo-containing egg of the same bird previously transferred by the same method using the same CTCs, but to which no therapeutic agent was administered. Similarly, if the effect of several therapeutic agents is studied, the effect will preferentially be evaluated by comparison of parameters after administration of a combination of therapeutic agents to an embryo-containing egg once transferred, in comparison with those determined in one or more other embryo-containing eggs of the same bird previously transferred by the same method using the same CTCs, but to which each of the therapeutic agents was administered individually.

[0059] According to another aspect, the present invention also provides a method for monitoring a patient or animal suffering from cancer, comprising the steps of: preparing a first avian embryo-containing egg as described above using the CTCs isolated from said patient or animal sample at T1 and studying the tumorigenesis of tumors arising in said first embryo-containing egg; preparing a second avian embryo-containing egg, as described above, using CTCs isolated from the same patient or animal sample at time T2, and studying the tumorigenesis of tumors developing in this second embryo-containing egg; Comparing the tumorigenesis of tumors developed in said first embryo-containing egg and said second embryo-containing egg. The present invention relates to a method comprising the steps of:

[0060] The present invention also relates to a method for screening a therapeutic agent for in vivo treatment of cancer, comprising: Implanting circulating tumor cells isolated from a sample of a patient or animal suffering from cancer at the level of the chorioallantoic membrane (CAM) of an avian embryo-containing egg, said embryo-containing egg having been previously incubated to a developmental stage corresponding to the formation of the CAM and corresponding to at least 8 days of chicken development, administering one or more candidate agents to said embryo-containing egg at least 12 hours after said transfer; and studying the effect of the administered therapeutic agent on tumorigenesis of tumors arising in implanted embryo-containing eggs. The method comprises the steps of:

[0061] "Candidate therapeutic agent" is understood to mean a chemical or biological therapeutic agent as defined above that may have anti-cancer activity, in particular that is potentially effective for treating the type of cancer that arose from the tumor-like cells implanted in said embryo-containing egg. The screening method according to the invention makes it possible to determine whether a candidate therapeutic agent has anti-cancer activity and whether it has anti-metastatic activity.

[0062] All the preferences and precisions mentioned above within the different methods also apply to the monitoring and screening methods according to the invention. EXAMPLES

[0063] The invention is illustrated below by the use of chicken embryo-containing eggs to expand human CTCs.

[0064] Preparation of CTCs CTCs to be transplanted into embryo-containing eggs are isolated from blood samples of patients suffering from metastatic lung, breast, or prostate cancer by size-based filtration of cells using the ISET method (Han Wei Hou et al., 2013) or by a microfluidic system using the ClearCell method (Laget S et al., 2017). The CTCs are then cryopreserved and thawed immediately prior to transplantation.

[0065] Tumor induction Fertilized eggs from White Leghorn chickens are incubated in dorsal position for 9-10 days at 37.5°C and 40% relative humidity. After this incubation period (E9 or E10), these eggs are ready to receive implantation. An opening is made in them while maintaining the integrity of the CAM. This integrity is compromised by drilling a small hole in the eggshell. Then, a hole is placed approximately 1 cm above the CAM. 2A window is cut into the egg. CTCs (5-2500 cells) are implanted by apposition onto the CAM, after which the eggs are incubated once more at 37.5 °C and 40% humidity. After implantation, in ovo tumors develop on the upper CAM. The eggs are observed a minimum of every 48 h to monitor their development.

[0066] A diagram of an embryo-containing egg with a CTC implantation site in the upper CAM is shown in Figure 1. An example of the experimental program is shown in Figure 2.

[0067] On developmental day 18 (E18), the top of the CAM is removed and washed with phosphate-buffered saline, then placed directly in paraformaldehyde (PFA) and fixed for 48 hours. The tumor is then carefully removed from the normal CAM tissue.

[0068] The collected tumors are then analyzed, specifically: Weighing to study tumor growth (Figure 2, first); for histological or genetic analysis (e.g., metastasis) ( Figure 2 , first time); Use for secondary transfer in a new batch of embryo-containing eggs on developmental day 9 for amplification (Figure 2, round 2). This process can be repeated as many times as necessary (regularly controlling the phenotype).

[0069] secondary transplant After 9 days of growth of the transplanted CTCs, the tumors that have developed are harvested and prepared to perform secondary transplants: the tumors are cut into small pieces and each piece is transplanted into the CAM of a new egg to obtain robust amplification of the CTCs.

[0070] Analysis of metastases Analysis of metastases can be performed either on a portion of the lower CAM (opposite the implantation site) or on embryonic tissue collected at the same time as the tumor and stored in a suitable manner. After extraction of total genomic DNA, detection of human cells in these samples can be performed by qPCR using specific primers for human Alu sequences (multicopy sequences well conserved in humans).

[0071] result The above experimental design was applied to CTCs isolated from the blood of patients suffering from lung cancer (#DA106, #DA107, #PA108, #CB110, #PC111, #VM109, #SH103, #CM105), CTCs isolated from the blood of patients suffering from breast cancer (#PS234, #SS226, #UR227, #LP229), and CTCs isolated from the blood of patients suffering from prostate cancer (#PT319, #BG320).

[0072] After 9 days on the CAM (E18), all patient CTC samples successfully engrafted onto the CAM and demonstrated tumor initiation. An in ovo picture of tumor growth after the first round of amplification is summarized in Figure 3.

[0073] Histological analysis by hematoxylin / eosin staining was performed on tumors derived from CTCs (#PS234) isolated from the blood of a patient with breast cancer, collected at the time of the first amplification, and then implanted into the upper CAM.

[0074] The results are summarized in FIG. 4 and indicate the presence of malignant tumor growth.

[0075] Finally, gene mutation analysis was performed on genomic DNA extracted from tumors derived from CTCs (#PS234) isolated from the blood of a patient suffering from lung cancer, collected at the time of the first amplification, by rapid sequencing using the "Next Generation Sequencing" (NGS) method, as reviewed by Yohe S & Thyagarajan B., using the human tumor markers TP53 (Mogi A & Kuwano H 2011) and KRAS (Gou LY et al., 2015) in 2017. DNA extraction was performed according to the method of the GeneJET FFPE DNA purification kit sold by ThermoFisher.

[0076] The results are summarized in Figure 5, which shows the presence of mutations in the human tumor markers TP53 (Figure 5A) and KRAS (Figure 5B) in two different tumors derived from the initial transplantation of CTCs (#SH103 and #CM105) from a patient suffering from lung cancer.

[0077] Tumors derived from the first transplant of CTCs isolated from the blood of a patient suffering from lung cancer were harvested at E18, minced, and then transplanted once more onto the CAM of a second set of embryo-containing eggs. In ovo images of tumor growth harvested at E18 after the second round of amplification are summarized in Figure 6 (A and B correspond to in ovo images of tumors on the CAM derived from two different secondary transplants).

[0078] Tumors derived from the first transplantation of CTCs (ClearCell Fx1 Microfluidics) isolated from the blood of a patient suffering from prostate cancer were collected at E18, cut into pieces and then transplanted once more onto the CAM of a second set of embryo-containing eggs. In ovo images of tumor growth are summarized in Figure 7. Image A represents a tumor obtained in ovo from CTCs after the primary transplantation. The two tumor initiation sites are indicated by arrows. Image B corresponds to that of an in ovo tumor on the CAM, derived from a secondary transplant generated from the tumor shown in A. Image C corresponds to the histological analysis of a section of the tumor shown in B, showing on one hand the CAM of the tissue and on the other the tumor tissue.

[0079] conclusion The above results show that the use of the avian embryo-containing egg model allows the in ovo expansion of CTCs isolated from patient samples in a more rapid and inexpensive manner compared to the prior art mouse model. Thus, the present invention allows the rapid construction of a biobank of CTCs, which in turn can be used for efficacy testing of anti-cancer therapies, monitoring and screening of novel agents capable of generating effective personalized treatments. References TIFF0007672335000001.tif202159TIFF0007672335000002.tif211159

Claims

1. A method for expanding human or animal circulating tumor cells (CTCs) isolated from a sample of a patient or animal suffering from cancer, comprising the steps of: a) preparing an avian embryo-containing egg model using a method for preparing an avian embryo-containing egg model that allows for the amplification of CTCs, the method comprising the steps of: a) transplanting uncultured CTCs isolated from said sample at the level of the chorioallantoic membrane (CAM) of an avian embryo-containing egg, said embryo-containing egg being at a developmental stage corresponding to the formation of the CAM and corresponding to at least 8 days of chicken development, and the amount of CTCs to be transplanted being between 5 and 5000; b) Harvesting tumors arising from the expanded CTCs The method comprising:

2. The method for expanding CTCs according to claim 1 , further comprising incubating the implanted embryo-containing egg for at least 12 hours in step a).

3. The method for expanding CTCs according to claim 1 or 2, further comprising: c) recovering CTCs from the tumor collected in step b).

4. The following steps: a) preparing a first avian embryo-containing egg model using a method for preparing an avian embryo-containing egg model that allows for the amplification of CTCs, comprising the steps of: a) transplanting uncultured human or animal circulating tumor cells (CTCs) isolated from a sample of a patient or animal suffering from cancer at the level of the chorioallantoic membrane (CAM) of an avian embryo-containing egg, said embryo-containing egg being at a developmental stage corresponding to the formation of the CAM and corresponding to at least 8 days of chicken development, and the amount of CTCs to be transplanted being between 5 and 5000; b) harvesting the tumors arising from the expanded CTCs; b1) implanting the tumor collected in step b) at the level of the chorioallantoic membrane (CAM) of a second avian embryo-containing egg, said second embryo-containing egg having been previously incubated until a developmental stage corresponding to the formation of the CAM and corresponding to at least 8 days of chicken development; b3) harvesting the tumors arising from the tumors implanted in step b1). The method for amplifying CTCs according to claim 1 or 2, comprising:

5. 5. The method for expanding CTCs according to claim 4, further comprising: b2) incubating the second embryo-containing egg transferred by step b1) for at least 12 hours.

6. The method for expanding CTCs according to claim 4 or 5, further comprising: c) recovering CTCs from the tumor collected in step b3).

7. 1. A method for assisting in determining the sensitivity of a patient suffering from cancer to one or more therapeutic agents, or for determining the sensitivity of a non-human animal suffering from cancer to one or more therapeutic agents, comprising: Expanding uncultured circulating tumor cells (CTCs) isolated from a sample of a patient or animal suffering from said cancer according to the method of any one of claims 1 to 6, implanting the tumor thus collected at the level of the chorioallantoic membrane (CAM) of a new avian embryo-containing egg, said new embryo-containing egg having been previously incubated until a developmental stage corresponding to the formation of the CAM and corresponding to at least 8 days of chicken development, the amount of CTCs to be implanted being between 5 and 5000; administering said therapeutic agent to said embryo-containing egg at least 12 hours after said transfer; studying the effect of the therapeutic agent administered thereby on the tumorigenesis of the harvested tumors that developed in the newly implanted embryo-containing eggs. The method of claim 1, comprising:

8. 8. A method for determining the sensitivity of a patient or animal suffering from cancer to one or more therapeutic agents as described in claim 7, wherein the study of tumorigenesis comprises the evaluation or measurement of different parameters such as tumor growth, metastatic invasion, angiogenesis, neovascularization, inflammation and / or tumor immune infiltration, toxicity towards the tumor and / or towards the whole embryo.

9. 1. A method for assisting in the monitoring of a patient suffering from cancer or a method for monitoring a non-human animal suffering from cancer, comprising: implanting uncultured human or animal circulating tumor cells (CTCs) isolated from said patient or animal sample at time T1 at the level of the chorioallantoic membrane (CAM) of an avian embryo-containing egg, preparing a first avian embryo-containing egg using a method for preparing an avian embryo-containing egg model that allows for the amplification of CTCs, the method comprising the steps of: said embryo-containing egg being at a developmental stage corresponding to the formation of CAMs and corresponding to at least 8 days of chicken development; and the amount of CTCs to be transplanted being between 5 and 5000; and studying the tumorigenesis of tumors arising in the first embryo-containing egg; preparing a second avian embryo-containing egg using a method for preparing an avian embryo-containing egg model that allows the amplification of CTCs, comprising the steps of: transplanting uncultured CTCs isolated from the same patient or animal sample at time T2 at the level of the chorioallantoic membrane (CAM) of an avian embryo-containing egg, said embryo-containing egg being at a developmental stage corresponding to the formation of the CAM and corresponding to at least 8 days of chicken development, and the amount of CTCs transplanted being between 5 and 5000; and studying the tumorigenesis of a tumor developing in this second embryo-containing egg; Comparing the tumorigenesis of tumors developed in said first embryo-containing egg and said second embryo-containing egg. The method comprising:

10. A method for screening a therapeutic agent for in vivo treatment of cancer, comprising: preparing an avian embryo-containing egg model using a method for preparing an avian embryo-containing egg model that allows for the amplification of CTCs, the method comprising the steps of: transplanting uncultured human or animal circulating tumor cells (CTCs) isolated from a sample of a patient or animal suffering from cancer at the level of the chorioallantoic membrane (CAM) of an avian embryo-containing egg, the embryo-containing egg being at a developmental stage corresponding to the formation of the CAM and corresponding to at least 8 days of chicken development, and the amount of CTCs to be transplanted being between 5 and 5000; administering one or more candidate agents to said embryo-containing egg at least 12 hours after transfer; and studying the effect of the administered therapeutic agent on tumorigenesis of tumors arising in the implanted embryo-containing eggs. The method comprising:

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