Patent Application
20250051703
The present invention relates to the field of establishment, preparation and use of shell-free (ex ovo) egg assays, and more particularly chorioallantoic membrane (CAM) assays, methods and their use. The invention further relates to techniques and equipment for egg embryo culturing and engineering and more particularly to avian egg embryo culturing and engineering. The present invention also relates to methods for treating Ewing sarcoma, rhabdomyosarcoma, and hepatoblastoma.
Preclinical cancer research ranges from in vitro studies that are inexpensive and not necessarily reflective of the tumor microenvironment to mouse studies that are better models but prohibitively expensive at scale. Previously, shell-free egg assays have been proposed using chicken eggs (see Fisher, “Chick Embryos in Shell-less Culture”, in Tested studies for laboratory teaching, pp. 105-115, Proceedings of the 5th Workshop/Conference of the Association for Biology Laboratory Education, Goldman, et all, cd., 1993; and PCT Publication WO 2005/033300 A1 to Sierra-Honigmann assigned to Cedars-Sanai Medical Center, published on Apr. 14, 2005). Assays employing shelled eggs, including chicken, duck, turkey, goose, quail, pheasant, grouse, ostrich, cmu, cassowary, and kiwi eggs, are also known (see PCT Publication No. WO 2010/054022 A1 to Chen, published on May 14, 2010, all of which are incorporated herein by reference) and employing various chorioallantoic membrane (CAM) assays.
The CAM is a membrane formed by the fusion of the choiron and allantois membrane on embryonic day 5-6 (see Lokman, et al., “Chick chorioallantoic membrane (CAM) assay as an in vivo model to study the effect of newly identified molecules on ovarian cancer invasion and metastasis”. Int J Mol Sci vol. 13, pp. 9959-70 (2012); Ainsworth, et al., “Developmental stages of the Japanese quail”, J Anat, vol. 216, pp. 3-15 (2010), both incorporated herein by reference). During normal development, the CAM will attach to the inside of the eggshell allowing respiration and calcium extraction for the growing embryo.
The typical methods for culturing avian embryos fall into two general categories: in ovo, whereby a small hole cut into the shell gives access to the embryo (see, for example, Handler, et al., “Studies on mortality in chick embryos resulting from implantation of whole blood and blood fractions from patients and animals with neoplastic disease”, Proc Natl Acad Sci USA, vol. 48, pp. 1549-73 (1962) incorporated herein by reference), and ex ovo with the embryo transferred to a container such as a cell culture plate and grown separate from the shell. The chick methods of in ovo incubation, while effective for chick survival, are time consuming and cannot be scaled because of the need for a skilled operator (see, Deryugina & Quigley, “Chick embryo chorioallantoic membrane model systems to study and visualize human tumor cell metastasis”, Histochem Cell Biol vol. 130, pp. 1119-30 (2008), incorporated herein by reference). For ex ovo approach, another group has reported a culture method for Japanese quail that is novel but also time intensive and unsuitable for automation in multi-well plates (see, Subauste, et al., “Evaluation of metastatic and angiogenic potentials of human colon carcinoma cells in chick embryo model systems”, Clin Exp Metastasis vol. 26, pp. 1033-47 (2009), and Kato, et al., “Culture System for Bobwhite Quail Embryos from the Blastoderm Stage to Hatching”, J Poultry Sci, pp. 155-58 (2013), both incorporated herein by reference).
Approaches to using the avian eggs and/or CAM assays have had preclinical drug administration (dosing) pharmacokinetic challenges, but in recent years the application of drugs into the quail or chick has been approached by topical and intravenous injection (Vargas, et al., “The chick embryo and its chorioallantoic membrane (CAM) for the in vivo evaluation of drug delivery systems”, Adv Drug Deliv. Rev. vol. 59, pp. 1162-76 (2007), incorporated herein by reference), and even intraperitoneally. Such methods again do not lend themselves to automation because of the need of a skilled operator to make the injections.
In addition, it has been found that topical application and even injection may not result in a precise concentration of delivered drug or compound. It has been found that in a topical application the drug may be diluted by an unknown and varying degree and further may spread through the embryo unevenly.
As avian embryotic cultures are not immunocompromised but are instead immune-tolerant, CAM experiments may be conducted for a variety of cancers. However, known CAM methods are very low-throughput as considerable technician expertise is required and there is significant technician-to-technician variability. Thus, the inventors are not aware of any medium-throughput or high-throughput CAM preparation methods, nor any medium-throughput or high-throughput CAM assays reported for analyzing either adult or pediatric cancers. Sec, e.g., CAM via chicken embryos available from INOVATION, La Tronche, France (https://www.inovotion.com/our-technology/a-unique-in-vivo-technology), which employs in ovo assays requiring significant manual manipulation and preparation. This is because for known ex ovo CAM assays, a rate-limiting step is caused by the technical difficulty to transfer of fertilized egg contents with an intact yolk to maintain the embryo viability. To date, such a step requires a skilled artisan to carefully make the transfer, and even with a trained technician this results in significant breakage and variability, all of which reduces throughput, speed and reliability.
CAM assays are also known to grow tumor cells on a 3D scaffold and to detect drug efficacy in, for example, Abraham, et al. “Evasion mechanisms to Igf1r inhibition in rhabdomyosarcoma”, Mol. Cancer There., vol. 10, pp. 697-707 (2011). In this paper, the fertilized quail eggs were washed, dried, sterilized, and incubated at 37.4° C. for 3 days (E3). Forceps were used to remove a small part of the egg shell and the contents of the egg were transferred to a well of a 6-well plate. At E6, 1×106 aleovar rhabdomyosarcoma cells grown on a 3D scaffold (3D Biotek) were added to the chorioallantoic membrane (CAM). Cells of this primary tumor culture also harbor a genetically engineered luciferase gene allowing their detection and quantification. The day following xenotransplantation, 20 μL of complete medium containing 10 μmol/L NVP-AEW541 or 100 μmol/L imatinib was added to the cells. Three days after adding the drug to the cells, 400 μL of 1.5 mg/mL luciferin diluted in PBS was added dropwise to the surface of the CAM. After 30 minutes, the quail embryo was imaged.
Chick embryos are well-established for CAM studies, and chick development is well-documented. Chick embryos typically require 20-21 days for full gestation, and a standardized cell culture plate may only contain 1-2 chicken embryos. Thus, it has now been recognized that the use of chicken embryos results in automation difficulties. However, the developmental stages of other avian species such as Japanese quail are also well-studied from early development to old age. See, e.g., Japanese quail (Coturnix japonica) as a laboratory animal model, Huss, et al., Lab Animal, vol. 37, No. 11, pp. 513-19, 2008, herein incorporated by reference in its entirety.
Thus, it has been found by the inventors that employing quail eggs in a CAM assay possess certain advantages over regular chicken eggs. For example, it has been found that quail eggs may further possess additional advantages such as being cheaper when purchased in bulk, well-sized to fit into a standard six-well dish/plate, and/or that quail eggs lead to easier automation. In a specific example, it has been found that typically only 1-2 chicken embryos would fit on a standard-sized cell culture plate, whereas 6 quail embryos fit on the same-sized plate.
Correlations between, for example quail embryos and chicken embryos are made by comparing literature sources of chicken embryo mass and blood volume with measured quail embryo growth over the same Hamburger and Hamilton stages (see, Mueller, et al., Sturkie's Avian Physiology, (Elsevier, New York, 2015), incorporated herein by reference), where the assumption is that the ratio of blood to body mass is the same during comparable growth stages.
Accordingly, there remains a need for more cost-effective methods and equipment for preparing, performing and using assays, especially CAM assays. There also remains a need for assays for testing drug concentration ranges and assays for rapid testing of ex ovo patient xenografts. There also exists a need for an assay comparable to murine in vivo xenografts, but which is faster and may be performed at a lower cost. There remains a need for an assay for assessing drug toxicity in eggs, and especially quail eggs. There remains a need for an assay employing transgenic eggs, and especially transgenic quail eggs, which allows rapid quantitative and qualitative testing using luminescent markers. There remains a need for an assay which quickly and cost-effectively detects the safety of drugs, and especially the kidney safety of drugs.
There further remains a need for an efficient method and equipment for providing a shell-free CAM assay, especially a shell-free quail egg CAM assay, ready for automated testing. There remains a need for a shell-free CAM assay validated with reporter systems based on imaging analysis, especially luminescent image analysis. There remains a need for an ex ovo assay, especially an ex ovo CAM assay for testing the efficacy and/or toxicity of single drug concentrations.
In an embodiment of the invention herein, an egg opener for a predetermined number of eggs contains a frame and a sliding blade holder. The frame contains a predetermined number of egg holders, each egg holder equipped for securing a single egg. The sliding blade holder contains a blade; or a predetermined number of blades; or wherein the predetermined number of blades is equal to the predetermined number of eggs. When the sliding blade holder slides along the frame, the blade cuts each egg within the predetermined number of egg holders.
Without intending to be limited by theory, it is believed that the egg opener herein may result in more efficient embryo transfer into the wells of a multi-well plate, may reduce damage to embryos during transfer, may reduce the time required to transfer embryos into the wells of a multi-well plate, may be automated, and may have other benefits as well.
In an embodiment of the invention herein, a method for preparing an ex ovo tumor xenograft contains the steps of: preparing an ex ovo CAM and adding a cell-infused media to the CAM, wherein the cell-infused media comprises tumor cells to form an ex ovo tumor xenograft.
It is also believed that the method for preparing an ex ovo tumor xenograft herein may be very effective, efficient, and/or may be automated to reduce the time required to prepare such xenografts.
In an embodiment of the invention herein, an assay for testing a therapeutic compound contains the steps of providing an ex ovo tumor xenograft as described herein, and adding a therapeutic compound to the CAM.
Without intending to be limited by theory it is believed that the assay for testing a therapeutic compound herein may lend itself to easy automation, may provide accurate and/or speedy results, and/or may be easy and/or cheap.
In an embodiment of the invention herein, a method for testing a therapeutic compound via an ex ovo quail egg xenograft assay comprising the steps of transferring an embryo to a well in a plate, subjecting the embryo to a CAM formation incubation of from about 60 hours to about 144 hours to develop a CAM, wherein the CAM comprises a CAM surface, culturing a plurality of cells, suspending the plurality of cells in a media to form a cell-infused media, adding the cell-infused media to a scaffold, creating a superficial injury on the CAM surface, placing a ring on the CAM surface, wherein the ring at least partially encloses the superficial injury, and placing the scaffold in the ring and on the CAM surface in contact with the superficial injury, where the scaffold fits within the ring. The method herein further contains the steps of combining a delivery media with a therapeutic compound, placing the delivery media in the ring and on top of the scaffold, subjecting the CAM to an experimental incubation, administering a marker to a marker location selected from the well, the embryo, the CAM and a combination thereof, and detecting the marker.
It is also believed that the method for testing a therapeutic compound herein may be relatively easy, quick, cheap, and/or lend itself to partial or full automation.
In an embodiment herein, a method for treating Ewing sarcoma includes the step of administering to a patient an effective amount of a VEGFR inhibitor, a poly-kinase inhibitor, a EGFR inhibitor, and a combination thereof; or cediranib, erlotinib, and a combination thereof.
Without intending to be limited by theory, it is believed that, based on the data presented herein, an effective amount of these compounds may form the basis for an effective treatment for Ewing sarcoma.
In an embodiment herein, a method for treating rhabdomyosarcoma comprising the step of administering to a patient an effective amount of a PI3K/mTOR inhibitor and a combination thereof; or BEZ235.
Without intending to be limited by theory, it is believed that, based on the data presented herein, an effective amount of these compounds may form the basis for an effective treatment for rhabdomyosarcoma.
In an embodiment herein, a method for treating hepatoblastoma comprising the step of administering to a patient an effective amount of a PLK inhibitor and a combination thereof; or PLK1, volasertib and a combination thereof.
Without intending to be limited by theory, it is believed that, based on the data presented herein, an effective amount of these compounds may form the basis for an effective treatment for hepatoblastoma.
The figures herein are for illustrative purposes only and are not necessarily drawn to scale. In the figures, any error bars indicate±the standard error of the mean unless otherwise indicated.
Unless otherwise specifically provided, all tests herein are conducted at standard conditions which include a room and testing temperature of 25° C., sea level (1 atm.) pressure, pH 7, 60% humidity, sanitary conditions, and all measurements are made in metric units. Furthermore, all percentages, ratios, etc. herein are by weight, unless specifically indicated otherwise. It is understood that unless otherwise specifically noted, the materials compounds, chemicals, etc. described herein are typically commodity items and/or industry-standard items available from a variety of suppliers worldwide.
As used herein, the term “plate” indicates any container used to contain the embryo, and is typically a standardized, standard-sized multi-well cell culture plate, such as, for example, a 6-well plate readily-available from multiple vendors around the world. Each well is typically shaped and sized so as to contain a single embryo. These plates are typically optically-transparent plastic or sometimes glass.
Typically most, if not all steps of the procedures and/or steps described herein will take place in a sterile environment unless otherwise specifically stated.
In an embodiment herein, we present an approach to increased CAM assay throughput in the preclinical prioritization of anti-cancer compounds via an automation-ready plate, such as a 6-well plate format.
Without intending to be limited by theory, it is believed that the CAM provides an excellent hypervascularized platform for growing a tumor xenograft. It is further believed that these tumor xenografts may then be used for quantitative and/or qualitative testing of therapeutic compound efficacy against the tumor xenograft.
Without intending to be limited by theory it is also believed that quail embryos provide faster results than, for example, chicken embryos. It is known that chicken embryos require from 20-21 days to gestate, while quail embryos only require from 16-20 days. Thus, as the developmental cycle may be about 25% faster, it has been found that the desired assay may be significantly accelerated when using quail embryos.
In addition, it is believed that the use of quail embryos in a CAM assay is much more space-efficient, as quail embryos are smaller, with 6 embryos fitting onto a standard 6-well cell culture plate, whereas only 1-2 chicken embryos may fit on a comparably-sized cell culture plate. Thus, it is believed that quail embryo CAMs more readily lend themselves to automation and efficient space use as compared to chicken CAMs, or CAMs employing larger eggs.
A schematic of an embodiment of a shell-free quail CAM assay herein is presented in
It is known in the industry that fertilized quail eggs (for example, eggs that have undergone full egg fertilization) may be purchased and stored under standard refrigeration (e.g., from about 1° C. to about 5° C.) for up to about 7 days, or 120 hours, before use. It is also known that these fertilized eggs do not start forming an embryo, growing and gestating until they are subject to an in ovo incubation as described herein.
While it is possible to include multiple embryos in a single well (see WO 2005/033300 A1 by Sierra-Honigmann, to Cedars-Sainai Medical Center, published on Apr. 14, 2005), in an embodiment herein each well contains a single embryo.
In an embodiment herein, the fertilized quail eggs are periodically rotated during the in ovo fertilization so as to reduce and/or prevent the embryo from attaching to the egg shell.
In Step 2, the fertilized egg yolk (hereinafter referred to as an “embryo”) and at least a portion of the egg white are transferred into a well in a plate; or multi-well plates; or a 6-well plate, to form a filled well in a plate. The plate herein may be a petri dish, or a standardized multi-well plate. Typically each of the wells of the plates will be circular and have a larger diameter than the yolk of the egg to be placed therein. In an embodiment herein, each well has a diameter that is from about 1.1 times to about 5 times; or from about 1.2 times to about 4 times; or from about 1.5 times to about 3 times larger than the diameter of the yolk. in an embodiment herein the wells are optically transparent; or the wells and the plates are optically transparent. In an embodiment herein, each well holds at least about 10 ml; or from about 10 ml to about 100 ml; or from about 12 ml to about 80 ml; or at least 15 ml; or from about 15 ml to about 50 ml.
In a typical ex ovo process known and used prior to the present invention, the transfer of the embryos to the well in the plate is a rate-limiting step requiring considerable expertise. Typically the time to process a single egg using conventional methods involves carefully cutting each egg open with scissors, manually removing the shell pieces with forceps, and carefully pouring the embryo into a dish or a well while avoiding puncture or breakage of the egg yolk, the loss of too much egg white, and/or damage to the embryo. It has been found that the results of this manual process is extremely variable (different amounts of white transferred, different viability ranges, speed variations, etc.), typically requiring from about 5 to about 15 minutes to fill a single 6 well plate, and requires intense focus and precision which wears on and is very stressful for the technician(s) performing the manual procedure. Furthermore, the transfer success rate varies significantly depending on the proficiency of the technician, with observed success rates of from about 30% to about 80%, where success is defined as successfully transferring an embryo without rupturing or otherwise damaging the embryo.
Accordingly, in an embodiment of the invention herein we describe herein a simple egg opener device to process multiple eggs (containing embryos) at a single time to allow the transfer of the embryo contained in the egg into a plate.
In an embodiment herein, the egg opener, 10, holds a predetermined number of eggs; or quail eggs. The egg opener contains a frame, 20, with a predetermined number of egg holders, 22, 22′, etc. each for securing a single egg, which in this embodiment are 6 round holes in an egg holder frame, 24.
In an embodiment herein, the egg holder frame, 22, may be removable, interchangeable, adjustable, etc. such that different-sized eggs and/or different numbers of eggs may be processed with the same egg opener merely by changing or adjusting the egg holder frame, 22. The egg holder frame, 24, is fixed to the frame, 20, during use and does not move relative to the frame, 20 during use. In an embodiment herein, the predetermined number of egg holders is from about 2 egg holders to about 24 egg holders; or from about 4 egg holders to about 12 egg holders; or from about 6 egg holders to about 10 egg holders; or about 6 egg holders. In an embodiment herein the predetermined number of eggs is the same as the predetermined number of egg holders.
Each of the egg holders in the predetermined number of egg holders may be each individually-sized to hold an egg selected from the group consisting of an avian egg; or a chicken egg, a quail egg and a combination thereof; or a quail egg; or a Japanese quail (Coturnix japonica) egg, although typically each of the egg holders in an egg holder frame will be sized to hold the same sized eggs.
In addition to the round holes shown in
The egg opener, 10, in
In an embodiment herein multiple egg holder frames and multiple corresponding plates may be present.
A blade, 32, is affixed to a sliding blade holder, 34. In an embodiment herein a plurality of blades are affixed to the sliding blade holder. In an embodiment herein, a single blade is affixed to the blade holder. In an embodiment herein the blade; or plurality of blades, is removable for replacement and/or cleaning.
In
In an embodiment herein the blade, 32, cuts through from about the bottom 10% to about the bottom 30% of the egg; or from about the bottom 15% to about the bottom 25% of the egg; or about the bottom 20% of the egg, as measured according to the height of the egg. Without intending to be limited by theory, it is believed that given the typical shape of a quail egg, this cutting off of the bottom of the egg balances the competing aims of providing a large enough hole in the eggshell (see
In the embodiment of
The alignment and location of the egg holders, 22, correspond to the alignment and location of the wells of a standardized multi-well cell culture plate; or a 6 well plate, such that when the egg opener, 10, of
Once the embryos have dropped into the 6-well plate, then the plate stand, 26, is slid in the direction of arrow B, and the 6-well plate (see
In order to minimize the potential damage to the embryo and/or rupture of the yolk caused by dropping an embryo into a well of the plate, it is desired to reduce the vertical distance between the blade, 32, and the top of the well (see
In a typical embryo transfer process using the egg opener, even an unskilled practitioner may transfer 6 embryos into a 6 well plate in less than 8 minutes; or from about 3 minutes to about 8 minutes, or even faster. Without intending to be limited by theory, it is believed that when employing the egg opener herein, a technician may be able to successfully transfer (e.g., the embryos are viable after transfer) a significantly greater number of embryos into a container or plate at a greater rate. For example, the inventors have found that when employing the device herein even a new technician may process 100 eggs in about 2 hours and 10 minutes, resulting in an average embryo transfer rate of about 1 embryo every 78 seconds, or a 6 well plate in about 7 minutes and 48 seconds. Generally, this is about half the time required using the conventional embryo transfer method.
In addition, it is believed that the transfer success rate may be significantly improved a well. For example, the inventors have found that when employing the device herein a technician may result in a transfer success rate of about 85%, as compared to an average success rate of about 55% when using the conventional embryo transfer method as shown in
In Step 3 (see
In Step 4 (see
In Step 5 (see
In Step 6 (see
In Step 7 (see
In Step 8 (see
Traditionally, CAM assays using avian embryos have applied drugs via topical or intravenous injection, which requires a skilled technician to avoid harm to the embryo and/or the CAM. However, in an embodiment of the present invention it has been found that a therapeutic compound may be applied to the CAM via a delivery media which may reduce the chance of embryo damage, provide a more precise dosage, provide a measured/constant diffusion into the scaffold, provide controllable therapeutic compound delivery, etc.
Thus, in Step 9 (see
The therapeutic compound herein may be any type of drug, small molecule, biological sample (e.g., an antibody, antibody-drug conjugate, a protac (proteolysis-targeting chimera), enzyme, protein, genetic material, vector, plasmid, virus, bacteria, amoeba, etc.), or other compound which the technician or others may wish to test against a particular type of cell or cells (e.g., against the ex ovo xenograft). The biological sample herein may be a wild type or engineered biological sample. It is understood that the therapeutic compound herein may include a single compound or a combination of compounds; may contain a full formulation, or a minimal formulation, may contain a theoretical or suspected therapeutic compound, or a proven therapeutic compound, etc. In an embodiment herein, the therapeutic compound is an anti-cancer therapeutic compound; or a suspected anti-cancer therapeutic compound. In an embodiment herein, the at least one cell is a tumor cell and the therapeutic compound is an anti-cancer therapeutic compound; or a suspected anti-cancer therapeutic compound. In an embodiment herein, the therapeutic compound is selected from the group of a PLK inhibitor, a PI3K/mTOR inhibitor, a VEGFR inhibitor, a poly-kinase inhibitor, an EGFR inhibitor, and a combination thereof; or volasertib, BEZ235, PLK1, cediranib, erlotinib, and a combination thereof.
In Step 10 (see
The ring diameter is typically larger than the scaffold diameter, and the ring height may be greater than the scaffold height, so as to allow the ring to hold a therapeutic compound as a drug depot (see Step 9) as the therapeutic compound diffuses into the scaffold. In an embodiment herein, the ring contains a mesh bottom with holes smaller than the particle size of the delivery media. In another embodiment, the ring lacks a bottom and thus the scaffold sits on the CAM, with the ring surrounding it. In an embodiment herein, the ring provides a barrier around and above the top edge of the scaffold to form the drug depot. In an embodiment herein, the ring is formed of plastic, silicone, metal and a combination thereof; or a plastic, silicone, gold, platinum and a combination thereof; or plastic, silicone, and a combination thereof.
In Step 11 (see
In an alternative embodiment herein, the delivery media; or the osmotic beads, (containing either the control or the therapeutic compound) are placed in the ring and on top of the scaffold, which is in turn located on the CAM. Without intending to be limited by theory, it is believed that the therapeutic compound passes from the delivery media to the scaffold and in turn into the CAM where it potentially affects the cells growing on the CAM.
In step 12 (see
In Step 13 (see
In Step 14 (see
In Step 15 (see
Alternatively, to the steps shown in
In an embodiment of the method and assay herein, a control is also conducted in parallel to the therapeutic compound. In the control, the therapeutic compound is typically replaced with a placebo and/or simply omitted from the media, CAM, etc. This allows a direct comparison between the activity of the therapeutic compound vs. when no therapeutic compound is present.
In an embodiment herein, no delivery media is used, and the therapeutic compound is delivered directly to the CAM.
In an embodiment herein, the cells; or tumor cells, are not suspended in a media, but are delivered directly to the CAM, for example after forming a plurality of cells.
An embodiment herein includes an assay for testing a therapeutic compound by providing an ex ovo tumor xenograft, especially as described herein, and adding a therapeutic compound to the CAM. It is understood that the steps and embodiments described in the method herein may also be steps or embodiments of the assay herein.
In an embodiment herein, the CAM assay is conducted with quail embryos and the endpoint significance is determined by an unpaired two-tailed t-test with Welch's correction. In such an embodiment, a p-value of less than 0.05 may be considered to be statistically significant.
Without intending to be limited by theory, it is believed that the use of the CAM assay herein may provide a cost-effective and time-efficient screening method to precede and/or minimize the scope and/or need for murine studies, especially for therapeutic compounds such as anti-cancer treatments and/or drug toxicity studies. We believe that the methods and CAM assay described herein may be applicable to a variety of biomedical research endeavors, such as, for example, single-dose therapeutic compound concentrations, therapeutic compound dose-response calculations for multiple concentrations, therapeutic compound dose-response curves, therapeutic compound toxicity tests, validation of marker systems, etc. Furthermore, it is shown that the methods herein may provide quantitative and/or qualitative results.
Because generation of patient-derived xenograft using immune-compromised host mice can take 2-7 or more months12, we tested whether flat sections of patient tumor would engraft on the CAM immune-tolerant platform (
The histology slides were examined by co-pathologist A.M and were found to have significantly more toxicity from the combination of cediranib and erlotinib than either drug alone. Synergistic toxicity was seen in the e10 quail embryo liver and kidney as shown in
Accordingly, it is believed that a VEGFR inhibitor, a poly-kinase inhibitor, a EGFR inhibitor, and a combination thereof; or cediranib, erlotinib, and a combination thereof, may be useful for the treatment of Ewing sarcoma. Thus, an embodiment of the invention herein is a method for treating Ewing sarcoma by administering to a patient an effective amount of a VEGFR inhibitor, a poly-kinase inhibitor, a EGFR inhibitor, and a combination thereof; or cediranib, erlotinib, and a combination thereof. Alternatively, an embodiment of the invention herein includes the use of an effective amount of a VEGFR inhibitor, a poly-kinase inhibitor, a EGFR inhibitor, and a combination thereof; or cediranib, erlotinib, and a combination thereof, in the manufacture of a medicament for the treatment of Ewing sarcoma.
U48484 mouse rhabdomyosarcoma cells were cultured, trypsinized and added to two different vials of hydrogel making a concentration of 106 cells per 50 μl. BEZ235 (cat #S1009, Selleck Chemicals, Houston, TX) in a solution of 0.1% DMF (cat #TS-20673, Thermo Fisher Scientific) in PBS was added to 106 U48484 mouse rhabdomyosarcoma cells mixed with 50 μl hydrogel for a final concentration of 500 nM BEZ235. For untreated eggs, 0.1% DMF in PBS was used as a control. 50 μl of cells/hydrogel/drug mixture were added to each scaffold and incubated for approximately 30-45 minutes at 37° C. and 100% humidity. As detailed above, a superficial injury was created on the chorioallantoic membrane and tumor module containing either drug or control was placed on top. Quail bearing tumor module models were incubated for 72 hours. Add the end of the incubation, 100 μl of PBS containing 1.5 mg of luciferin-d (cat #122799, PerkinElmer) was added to the 3D scaffold, incubated for 10 minutes in the dark, and bioluminescence was measured using a Fluorchem instrument (ProteinSimple, San Jose, CA). The quail were imaged with an 8 minute exposure for total light emission.
Accordingly, it is believed that a PI3K/mTOR inhibitor and a combination thereof; or BEZ235, may be useful for the treatment of rhabdomyosarcoma. Thus, an embodiment of the invention herein is a method for treating rhabdomyosarcoma by administering to a patient an effective amount of a PI3K/mTOR inhibitor and a combination thereof; or BEZ235. Alternatively, an embodiment of the invention herein includes the use of an effective amount of a PI3K/mTOR inhibitor and a combination thereof; or BEZ235, in the manufacture of a medicament for the treatment of rhabdomyosarcoma.
A PLK inhibitor (volasertib) was selected for testing against a range of hepatoblastoma cell lines given that PLK1 is a proposed therapeutic target. The canonical hepatoblastoma cell line HepG2 and patient-derived xenograft (PDX) explanted cell lines HB243 and HB282 were selected as contemporary, robust patient-derived comparators. Volasertib was tested both in vitro and ex ovo against the cell lines across a concentration range. Previous in vitro studies and our own results show that HB282 and HepG2 both were least sensitive to volasertib (IC_50 values 916 nM
Accordingly, it is believed that a PLK inhibitor and a combination thereof; or volasertib, may be useful for the treatment of hepatoblastoma. Thus, an embodiment of the invention herein is a method for treating hepatoblastoma by administering to a patient an effective amount of a PKI inhibitor and a combination thereof; or volasertib. Alternatively, an embodiment of the invention herein includes the use of an effective amount of a PKI inhibitor and a combination thereof; or volasertib, in the manufacture of a medicament for the treatment of hepatoblastoma.
The tumor modules for dose response assay are generated as described above but with 5×105 cells per 50 μl. Drug was dissolved in DMSO for all levels to a final tumor module concentration of vehicle control, 0.3 μM, 3 μM, or 30 μM with n=6. P-10 beads (catalog #1504144, Bio-Rad, Hercules, CA, USA) are soaked in PBS at the concentration of the modules for four hours at room temperature. Approximately 50 μl of bead solution is added to a 9.5×1.5 mm plastic ring placed on top of the tumor module forming a drug depot. The drug depot provided a constant source of drug keeping the tumor module at a constant concentration despite drug leaving the module for the quail, as shown in
Validation Assays with Murine and Human Cell Lines
Validation assays are conducted to correlate murine and human cell lines across a range of biologicals and small molecule drugs for pediatric and adult cell lines. B16F10 is a murine melanoma cell line constitutively expressing firefly luciferase (available as cat #BW124734 from Perkin-Elmer, Akron, Ohio USA), which is used to form a CAM xenograft as per the invention herein with the difference of being an adult-type cancer cell line, and radiance reflecting cell viability as impaired by drug. In
As can be seen in
Validation Assay with Human Breast Cancer Cell Line.
BT474 is a human breast cancer cell line constitutively expressing firefly luciferase (available as catalog #SC-1232 from Cellomics Technology, Halethorpe, MD USA), which is used to form a CAM xenograft as per the invention herein but using an adult-type of cancer cell line instead of a pediatric cell line, most similar to example 5.
As can be seen in
Validation Assay with Human Diffuse Intrinsic Pontine Glioma (DIPG) Cancer Cell Line.
SF8628 is a pediatric human diffuse intrinsic pontine glioma (DIPG) cancer cell line transfected with a non-replicative lentivirus to constitutively expressing firefly luciferase (available as catalog #SCC127 from Sigma-Aldrich, St Louis, MO, USA), which is used to form a CAM xenograft as per the invention herein, which like example 4 shows the use of the assay for a pediatric brain tumor cell line.
As can be seen in
Validation Assay with Human Hepatoblastoma Cell Line.
HepG2 is a human pediatric hepatoblastoma cell line transfected with a non-replicative lentivirus to constitutively express firefly luciferase (available as catalog #8065 from ATCC, Manassas, VA, USA), which is used to form a CAM xenograft as per the invention herein most similar to example 4.
As can be seen in
Validation Assay with Murine Rhabdomyosarcoma Cell Line.
u48484 is a murine rhabdomyosarcoma cell line constitutively expressing firefly luciferase (available from the originating laboratory as described in Aslam et al, PDGFRβ reverses EphB4 signaling in alveolar rhabdomyosarcoma, Proc Natl Acad Sci USA. 2014 Apr. 29; 111 (17): 6383-8. doi: 10.1073/pnas. 1403608111; PMID 24733895), which is used to form a CAM xenograft as per the invention herein except being a non-human tumor cell line.
As can be seen in
Non-limiting embodiments of the invention herein include:
Embodiment 1) An egg opener for a predetermined number of eggs comprising:
Embodiment 2) The egg opener according to embodiment 1, wherein the predetermined number of eggs is from about 2 eggs to about 24 eggs; or from about 4 eggs to about 12 eggs; or from about 6 eggs to about 10 eggs; or about 6 eggs.
Embodiment 3) The egg opener according to any one of the previous embodiments, wherein the predetermined number of egg holders are each sized to hold an egg selected from the group consisting of an avian egg; or a chicken egg, a quail egg and a combination thereof; or a quail egg; or a Japanese quail (Coturnix japonica) egg.
Embodiment 4) The egg opener according to any one of the previous embodiments, further comprising a standardized multi-well cell culture plate affixed to the egg opener, wherein the alignment and location of the predetermined number of egg holders corresponds to the alignment and location of the wells of the standardized multi-well cell culture plate, and wherein when the blade cuts the eggs the egg contents transfer to the plurality of wells.
Embodiment 5) The egg opener according to any of the previous embodiments, wherein when the blade cuts each egg, the sliding blade holder collects the egg shell fragments.
Embodiment 6) The egg opener according to embodiments 4, wherein the standardized multi-well plate is removably-affixed to the egg opener.
Embodiment 7) A method for preparing an ex ovo tumor xenograft comprising the steps of:
Embodiment 8) The method for preparing an ex ovo tumor xenograft according to embodiments 7, further comprising the step of adding the cell-infused media to a scaffold prior to adding the cell-infused media to the CAM.
Embodiment 9) The method for preparing an ex ovo tumor xenograft according to any one of embodiments 7 to 8, further comprising the step of creating a superficial injury to the CAM and placing the cell-infused media in contact with the superficial injury.
Embodiment 10) The method for preparing an ex ovo tumor xenograft according to any one of embodiments 7 to 9, further comprising an experimental incubation wherein the CAM is incubated for from 0 hours to about 144 hours after adding the cell-infused media to the CAM.
Embodiment 11) The method for preparing an ex ovo tumor xenograft according to any one of embodiments 7 to 10, wherein the tumor cells are suspended in a media; or a gel; or a hydrogel and a combination thereof, to form the cell-infused media.
Embodiment 12) An assay for testing a therapeutic compound comprising the steps of:
Embodiment 13) The assay for testing a therapeutic compound according to embodiments 12, further comprising the steps of:
Embodiment 14) The assay for testing a therapeutic compound according to any one of embodiments 12 or 13, wherein the therapeutic compound is provided in a delivery media; or wherein the delivery media is selected form the group consisting of a gel, a solid, a liquid and a combination thereof; or a gel, a solid and a combination thereof; or a solid; or a bead; or an osmotic bead.
Embodiment 15) The assay for testing a therapeutic compound according to any one of embodiments 12 to 14, comprising an experimental incubation step wherein the CAM is incubated for from 0 hours to about 144 hours after adding the cell-infused media to the CAM.
Embodiment 16) The assay for testing a therapeutic compound according to any one of embodiments 12 to 15, wherein a plurality of different therapeutic compound concentrations are tested on a plurality of CAMs.
Embodiment 17) The assay for testing a therapeutic compound according to any one of embodiments 12 to 16 further comprising the steps of:
Embodiment 18) A method for testing a therapeutic compound via an ex ovo quail egg xenograft assay comprising the steps of:
Embodiment 19) The method for testing a therapeutic compound via an ex ovo quail egg xenograft assay according to embodiment 18, wherein the plurality of cells are a tumor cell; or a liver carcinoma cell; or a hepatoblastoma cell; or a HepG2 human liver hepatoblastoma cancer cell, (ATCC HB-8065); or a Ewing sarcoma cell, or a HB234 cell; or a HB282 cell; or a rhabdomyosarcoma cell; or a U48484 (u48484) rhabdomyosarcoma cell; or a SF8628 cell; or a BT474 cell; or a B16F10 cell.
Embodiment 20) The method for testing a therapeutic compound via an ex ovo quail egg xenograft assay according to any one of embodiments 18 to 19, wherein the media is a gel; or a hydrogel.
Embodiment 21) The method for testing a therapeutic compound via an ex ovo quail egg xenograft assay according to any one of embodiments 18 to 20, wherein the experimental incubation is from about 0 hours to about 144 hours.
Embodiment 22) The method for testing a therapeutic compound via an ex ovo quail egg xenograft assay according to any one of embodiments 18 to 21, wherein the marker is a luminescent marker, a radioactive marker, a fluorescent marker, and a combination thereof; a luminescent marker, a fluorescent marker; and a combination thereof; or a bioluminescent marker; or a fluorescent marker.
Embodiment 23) The method for testing a therapeutic compound via an ex ovo quail egg xenograft assay according to any one of embodiments 18 to 22, further comprising the step of analyzing the experimental data.
Embodiment 24) A method for treating Ewing sarcoma comprising the step of administering to a patient an effective amount of a VEGFR inhibitor, a poly-kinase inhibitor, a EGFR inhibitor, and a combination thereof; or cediranib, erlotinib, and a combination thereof.
Embodiment 25) A method for treating rhabdomyosarcoma comprising the step of administering to a patient an effective amount of a PI3K/mTOR inhibitor and a combination thereof; or BEZ235.
Embodiment 26) A method for treating hepatoblastoma comprising the step of administering to a patient an effective amount of a PLK inhibitor and a combination thereof; or PLK1, volasertib and a combination thereof.
It should be understood that the above only illustrates and describes representative examples whereby the present invention may be carried out, and that modifications and/or alterations may be made thereto without departing from the spirit of the invention.
It should also be understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately, or in any suitable subcombination.
All references specifically cited herein are hereby incorporated by reference in their entireties. However, the citation or incorporation of such a reference is not necessarily an admission as to its appropriateness, citability, and/or availability as prior art to/against the present invention.
The below is a reproduction of a research paper submitted for publication by the inventors. This research paper was published on Dec. 2, 2021, and may be cited as Rasmussen et al, Preclinical therapeutics ex ovo quail eggs as a biomimetic automation-ready xenograft platform, Sci Rep. 2021 Dec. 2; 11 (1): 23302. doi: 10.1038/s41598-021-02509-3; PMID 34857796
Funding for this work was supported by the Macy Easom Cancer Research Foundation, as well as The Foundation for Addie's Research, Owls for Avery Foundation, Rutledge Foundation, the Super Sam Foundation, the Go4TheGoal Foundation and the Sam Day Foundation.
Histology for this work was done by the OHSU Histopathology Shared Resource.
Preclinical cancer research ranges from in vitro studies that are inexpensive and not necessarily reflective of the tumor microenvironment to mouse studies that are better models but prohibitively expensive at scale. Chorioallantoic membrane (CAM) assays utilizing Japanese quail (Coturnix japonica) are a cost-effective screening method to precede and minimize the scope of murine studies for anti-cancer efficacy and drug toxicity. To increase the throughput of CAM assays we have built and optimized an 11-day platform for processing up to 200 quail eggs per screening to evaluate drug efficacy and drug toxicity caused by a therapeutic. We demonstrate ex ovo concordance with murine in vivo studies, even when the in vitro and in vivo studies diverge, suggesting a role for this quail shell-free CAM xenograft assay in the validation of new anti-cancer agents.
Pediatric cancer has historically limited new drug development as demonstrated by only 10 new agents earning primary childhood cancer FDA approval since 1978. To develop new therapies for rare disease, a cost-effective workflow from basic science target identification to preclinical research and then to clinical investigation is needed. Current preclinical research approaches move from in vitro studies to in vivo murine models; however, most of the time drug response data obtained from in vitro assays fail to be confirmed in vivo. As mouse studies are sometimes prohibitively expensive and time-consuming, taking tens of thousands of dollars and 10 or more weeks to complete, it is crucial to develop innovative cost and time-effective processes to improve the selection of anti-cancer agents to be prioritized for preclinical mouse studies Here we propose a re-examined and optimized the shell-free quail chorioallantoic membrane assay (CAM) as a precursor to mouse preclinical studies1,2.
CAM assays have traditionally utilized chicken (chick) or quail eggs, with chick being used more commonly. Chick CAM models have been employed for the study of angiogenesis, tumor growth and metastasis3-5. The CAM is a membrane formed by the fusion of the chorion and allantois membrane on embryonic day 5-6 (e5-6)6. The membrane will attach to the inside of the eggshell allowing respiration and calcium extraction for the growing embryo. The methods for culturing embryos fall into two categories: in ovo, whereby a small hole cut into the shell gives access to the embryo (for example, the studies performed by Sidney Farber in 19625), or ex ovo with the embryo transferred to a cell culture plate and grown separate from the shell. The chick methods of in ovo incubation, while effective for chick survival, are time consuming and cannot be scaled because of the need for a skilled operator7. For ex ovo approach, another group has reported a culture method for Japanese quail that is novel but also time intensive and unsuitable for automation in multi-well plates4,8. Parenthetically, too, the Japanese quail (Coturnix japonica) genome was sequenced in 2016 and is publicly available.
Approaches to using the avian eggs and/or CAM assays have had preclinical drug administration (dosing) pharmacokinetic challenges, but in recent years the application of drugs into the quail or chick has been approached by topical and intravenous injection9. To circumvent systemic drug administration, drug can be admixed with tumor cells in an extracellular matrix that is applied to the quail CAM1,3.
CAM experiments have been conducted on a variety of cancers, but to our knowledge no medium- or high-throughput methods have been reported for either adult or pediatric cancers. Herein, we present an approach to increased throughput of CAM assays in the preclinical prioritization of anti-cancer compounds via an automation-ready 6-well plate format.
A schematic of the shell-free quail CAM assay is presented in
We next confirmed bioluminescence of tumor cells using a commercially-available luciferase-red fluorescent protein (RFP) lentivirus reporter system (
We initially validated our system using the dual PI3K/mTOR inhibitor BEZ235 was tested on the rhabdomyosarcoma cell culture U484842. A 3D scaffold (
A PLK inhibitor (volasertib) was selected for testing against a range of hepatoblastoma cell lines given that PLK1 is a proposed therapeutic target10. The canonical hepatoblastoma cell line HepG2 and patient-derived xenograft (PDX) explanted cell lines HB243 and HB282 were selected as contemporary, robust patient-derived comparators11. Volasertib was tested both in vitro and ex ovo against the cell lines across a concentration range. Previous in vitro studies10 and our own results show that HB282 and HepG2 both were least sensitive to volasertib (IC50 values 916 nM
In cell line-based mouse xenograft studies, HB243 and HepG2 had different responses to volasertib in vivo. HB243 caused a statistically significant reduction in growth relative to the control group as shown in
Because generation of patient-derived xenograft using immune-compromised host mice can take 2-7 or more months12, we tested whether flat sections of patient tumor would engraft on the CAM immune-tolerant platform (
The histology slides were examined by co-pathologist A.M and were found to have significantly more toxicity from the combination of cediranib and erlotinib than either drug alone. Synergistic toxicity was seen in the e10 quail embryo liver and kidney as shown in
Herein we report methods for reliably and reproducibly performing ex ovo drug testing in shell-free quail CAM assay xenografts. We believe this platform brings us close to automation through mechanical systems for transferring egg contents to multi-well plates and we demonstrate the feasibility of single concentration xenograft testing, or xenograft testing across a concentration range (e.g., the ex ovo IC_50). When culturing the quail ex ovo we noticed a majority of the die off between e4 and e7 most likely due to the shock of transferring the embryo to the six well plate. At e7 the chorioallantoic membrane had fully formed and providing a capillary rich surface to support the tumor module. We tested volasertib in mouse xenografts and compared the in vivo results to the ex ovo quail xenograft results and observed that HepG2 continued to be resistant in concordance with the ex ovo experiments and in contrast to the in vitro experiments. HB243 had a statistically significant reduction in growth in concordance to both the sensitivity of HB243 to volasertib in the ex ovo and in vitro experiments. The resistance of HepG2 to volasertib in vivo and ex ovo but not in vitro deserves further analysis. We conducted RNA sequencing that could not discern ABC transporters overexpression as the cause of resistance. Our validation studies were conducted solely for hepatoblastoma, but parallel remain to be done for other pediatric and adult cancers. The readiness in which patient xenografts engraft to the CAM is an exciting opportunity for further research.
Future directions will include addressing the pharmacokinetic considerations for efficacy and toxicity models in lieu of our described approach of hydrogel-based tumor modules as a single compartment model. Given the small blood volumes of the quail CAM, and the changing blood volumes from e7 to e11, serial micro-sampling approaches will require careful but worthwhile optimizations.
All experiments were conducted in accordance with Children's Cancer Therapy Development Institute policies and all relevant guidelines. Coturnix japonica eggs were purchased from Boyd's Bird Company (Eagle Creek, OR) and PurelyPoultry (Fremont, WI), stored at 4° C. for 120 hours and then incubated at 37° C. and 70% humidity for approximately 72 hours. Quail eggs were opened by our mechanical device (
All cell lines were obtained as de-identified samples. HepG2 human liver carcinoma cells (ATCC, HB-8065) were transfected with lentiviral particles containing RFP, luciferase, and neomycin resistance following manufacturer's instructions (cat #LVP677, Gentarget, San Diego, CA). Reporter-transfected cells were purified using 800 nM G418 antibiotic selection 24 hours, flow sorted, then antibiotic selected again with the resulting cell line stably expressing RFP and luciferase. Cells were maintained in DMEM (cat #11990573, Thermo Fisher Scientific, Waltham, MA) with 10% FBS (Thermo Fisher Scientific, cat #10437036) and 1% penicillin-streptomycin (cat #15140122, Thermo Fisher Scientific). HB282 was received from co-author Stefano Cairo [Xentech] and transfected with a lentiviral particle containing RFP, luciferase, and puromycin resistance following manufacturer's instructions (cat #LVP674, Gentarget). HB282 followed the previously listed selection process but with a puromycin (cat #73342, Stemcell Technologies, Cambridge, MA) concentration of 2 μg/ml. HB282 was cultured in ADMEM, 10% FBS, 1% penicillin-streptomycin, 1% L-glutamine. We received HB243 transfected with GFP and luciferase from co-author Stefano Cairo [Xentech]. Previously characterized U48484 murine alveolar rhabdomyosarcoma (aRMS) cells which stably express a luciferase reporter transgene were maintained in DMEM, 10% FBS, and 1% penicillin-streptomycin 2.
All experiments were conducted in accordance with Children's Cancer Therapy Development Institute policies and all relevant guidelines. To generate a standard curve for luminescence, 7 different cell densities ranging from 0 to 4×106 of HepG2Glo were suspended in Hydrogel-c (cat #GS313, ESI-BIO, Alameda, CA) and 50 μl were added to 9.5 mm diameter sterilized fiberglass 3D mesh in
Patient-Derived Xenograft onto the CAM
A diagram of the procedure is presented in
All experiments were conducted in accordance with Children's Cancer Therapy Development Institute policies and all relevant guidelines. In order to develop pharmacokinetic approximations for the quail we compared literature sources for chicken embryo mass and blood volume growth to our measured quail embryo growth over the same Hamburger and Hamilton stages
All experiments were conducted in accordance with Children's Cancer Therapy Development Institute policies and all relevant guidelines. U48484 mouse rhabdomyosarcoma cells were cultured, trypsinized and added to two different vials of hydrogel making a concentration of 106 cells per 50 μl. BEZ235 (cat #S1009, Selleck Chemicals, Houston, TX) in a solution of 0.1% DMF (cat #TS-20673, Thermo Fisher Scientific) in PBS was added to 106 U48484 mouse rhabdomyosarcoma cells mixed with 50 μl hydrogel for a final concentration of 500 nM BEZ235. For untreated eggs, 0.1% DMF in PBS was used as a control. 50 μl of cells/hydrogel/drug mixture were added to each scaffold and incubated for approximately 30-45 minutes at 37° C. and 100% humidity. As detailed above, a superficial injury was created on the chorioallantoic membrane and tumor module containing either drug or control was placed on top. Quail bearing tumor module models were incubated for 72 hours. Add the end of the incubation, 100 μl of PBS containing 1.5 mg of luciferin-d (cat #122799, Perkin Elmer) was added to the 3D scaffold, incubated for 10 minutes in the dark, and bioluminescence was measured using a Fluorchem instrument (Protein Simple, San Jose, CA). The quail were imaged with an 8 minute exposure for total light emission.
All experiments were conducted in accordance with Children's Cancer Therapy Development Institute policies and all relevant guidelines. The tumor modules for dose response assay were generated as described above but with 5×105 cells per 50 μl. Drug was dissolved in DMSO for all levels to a final tumor module concentration of vehicle control, 0.3 μM, 3 μM, or 30 μM with n=6. P-10 beads (cat #1504144, Bio-Rad, Hercules, CA) were soaked in PBS at the concentration of the modules for four hours at room temperature. Approximately 50 μl of bead solution was added to a 9.5×1.5 mm plastic ring placed on top of the tumor module forming a drug depot. The drug depot provided a constant source of drug keeping the tumor module at a constant concentration despite drug leaving the module for the quail, as shown in
All studies in mice were performed after receiving approval from the institutional animal care and use committee (IACUC) at Children's Cancer Therapy Development Institute and in accordance with ARRIVE Guidelines. HepG2 and HB243 were suspended in Matrigel and injected into n=10 eight week-old female nod scid gamma mice per cell line xenograft (Charles River, Hollister, CA, NOD.CB17-Prkdescid/NCrCrl) with 2×106 cells per 100 μl injection. The dosing schedule shown in
All experiments were conducted in accordance with Children's Cancer Therapy Development Institute policies and all relevant guidelines. Cediranib (Cat #S1017, Selleck Chemicals LLC, Houston, TX) and erlotinib (Cat #S7786) were purchased from Selleck Chemicals and reconstituted in dimethyl sulfoxide (DMSO) following the manufacturers recommendations and diluted to 10 mM stock concentration.
Fertilized Japanese quail eggs were incubated and plated as described previously. Quail were allowed to grow ex ovo in six-well plates until the quail had passed the patterning phase (e8 based on plating date). Each experimental arm was assigned n=4 viable quail and treated with one of four experimental conditions: vehicle, cediranib, ertoltinib, and cediranib+erlotinib. Dosages provided to quail were based on maximum clinically-achievable serum concentrations (Cmax) in human patients, specifically 42 ng/ml for cediranib13 and 1.3 μg/mL for erlotinib14. Stock concentrations were diluted in phosphate-buffered saline (PBS) to the respective target concentrations and to a final volume of 25 μL per agent. DMSO for vehicle was set at the DMSO volume used in the cediranib+erlotinib combination (8.4 μL DMSO).
Vehicle and diluted agents were subsequently applied dropwise to the quail chorioallantoic membrane. Quail were photographed at 0 hours, 24 hours, and 48 hours. Remaining viable quail (n=4 vehicle, n=3 cediranib, n=3 erlotinib, n=4 combination) were sacrificed 48 hours after dosing and fixed in 10% formalin for 24 hours. Fixed quail were transported to the OHSU Histology core, paraffin embedded, sectioned in coronal orientation, and stained with hematoxylin and eosin. Stained images were analyzed by pathologist co-author A.M. for kidney and liver histopathology looking for signs of normal versus abnormal development (
Each cell line was grown to 80% confluency, trypsinized, and snap frozen. RNA was extracted and sequenced by Beijing Genomics Institute (BGI, San Jose, CA). The quality of RNA prior to extraction was adequate for each cell line (DV<200%). HiSeq 4000 was used for paired-end sequencing with 40 million reads for RNA. Raw FASTQ sequencing files were run through our in-house computational pipeline.
For the murine in vivo xenograft study survival analysis, the tumor endpoint volumes for time-to-event (TTE) analysis were set at 0.75 cc and were collected to 1.5 cc. TTE was defined in days by selecting the day in which the tumor volume equaled or surpassed 0.75 cc. Animals that did not reach endpoint volume were assigned a TTE of 21 days for the HB243 analysis and a TTE of 12 days for the G2 analysis. The Kaplan-Meier survival plot represents the percentage of animals surviving at different time points during the study. These percentages were generated from the TTE data using GraphPad Prism 9.0 software (Graphpad Software, San Diego, CA, https://www.graphpad.com/scientific-software/prism/). Survival curve comparisons were analyzed using the Mantel-Cox and Gehan-Breslow-Wilcox tests (95% CI) through Graph Pad Prism 9.0 software. For the quail xenograft assay, significance was determined by an unpaired two-tailed t-test with Welch's correction and a p-value less than 0.05 was considered statistically significant. Error bars represent±standard error of the mean (SEM).
Funding for this work was supported by the Macy Easom Cancer Research Foundation, as well as The Foundation for Addie's Research, Owls for Avery Foundation, Rutledge Foundation, the Super Sam Foundation, the Go4TheGoal Foundation and the Sam Day Foundation.
Histology for this work was done by the OHSU Histopathology Shared Resource.
Overall conception and design: CK
Analysis and interpretation of experimental data: CK, SVR, LHP, AM, SR
Development of experimental methodology: CK, SVR, NEB
Provision of experimental reagents: SC, SVR
Acquisition of experimental data: SVR, NEB, SR
Writing, review and/or revision: CK, SVR, SR, SC
Study supervision: CK
The authors declare no conflict of interests.
The present application is a national phase entry under 35 U.S.C. § 371 of International Application No. PCT/US2022/046097, filed Oct. 7, 2022, which designates the United States of America, which claims priority to U.S. Provisional Application No. 63/253,261, filed Oct. 7, 2021, the entire disclosures of each of these applications are hereby incorporated by reference in their entireties and for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/046097 | 10/7/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63253261 | Oct 2021 | US |
