Patent Application
20220243160
Many in vitro cellular studies are based on cell monolayer cultures and cannot satisfactorily mimic the complex three-dimensional environments and multi-component/constituent structure of biological tissue, including organs, tumors, and soft tissue. The status quo as it pertains to develop new anticancer drugs is to use monolayer (2D) cell culture or animal models in preclinical phase in order to assess efficacy, distribution and toxicity of the compounds. However, 2D cell culture does not recapitulate cell-cell interactions, cell-matrix interactions, nutrient and oxygen gradients, or cell polarity and, in addition to all ethical problems, animal models present important differences with human physiology. Hence, these models appear to be too limited and explain the important rate of failure of drug candidate in clinical phase while rising up the development cost.
Although some prior art multi-culture systems have been developed, they still suffer from the fundamental disadvantage of not reproducing the complete architecture of the modeled biological system. For example, the tumor microenvironment (TME) has a high impact on cancer cell radiation response and, ultimately, patient survival after radiation treatment. Accordingly, there is a need in the art for cell culture methods and systems that better reflect the actual 3-D properties, both physical and biological, of a biological tissue. In the context of cancer cell treatment, improved cancer cell culture systems that address these issues would be beneficial for a range of therapeutic and treatment applications.
Provided herein are methods and systems that address the above problems by utilizing a modular microfluidic device for simulating in vivo conditions of a biological system. The modular microfluidic device may be configured to facilitate growth of the one or more 3D structures of cultured biological cells along all three dimensional axes. The methods and systems provided herein are compatible with a range of tissue cells, depending on the application of interest, such as cellular components of an organ, cancer cells of a tumor, cells of the vasculature, such as endothelial cells and smooth muscle cells, and combinations thereof. Any of the methods and systems provided herein may further comprise extracellular matrix, such as collagen, enzymes, and glycoproteins, that provide structural and biochemical support of surrounding cells.
In one embodiment, a modular microfluidic device for simulating in vivo dynamic conditions of a biological system comprises a first perfusion chamber having an inlet and an outlet, a second perfusion chamber having an inlet and an outlet, a well configured to hold one or more 3D structures of cultured biological cells and a lid. The well may be in fluid communication with the first and second perfusion chambers. The device may include a lid to seal the well while fluid is flowing through the first and/or second perfusion chambers. A first porous membrane may be disposed between the first perfusion chamber and the well. A second porous membrane may be disposed between the second perfusion chamber and the well. The well may be configured to facilitate growth of the one or more 3D structures of cultured biological cells along all three dimensional axes.
In one embodiment, a modular microfluidic device for simulating in vivo conditions of a biological system may include a first channel member having a first perfusion chamber formed therein, a second channel member having a second perfusion chamber formed therein, and a central member disposed between the first channel member and the second channel member. The first perfusion chamber may have an inlet and an outlet. The second perfusion chamber may have an inlet and an outlet. A first porous membrane may be disposed between the first perfusion chamber and the at least one well. A second porous membrane may be disposed between the second perfusion chamber and the at least one well. The central member may have a well formed therein, wherein the well is configured to facilitate growth of the one or more 3D structures of cultured biological cells along all three dimensional axes.
In one embodiment, the device may include a 3D structure of cultured biological cells disposed in the well of the central member. In one embodiment, the at least one well includes a first orifice in fluid communication with the first perfusion chamber via the first porous membrane, and a second orifice in fluid communication with the second perfusion chamber via the second porous membrane.
In one embodiment, an array of wells may be formed in the central member. In one embodiment, a first layer of living cells may be disposed on a surface of the first porous membrane. In one embodiment, the first layer of living cells comprises endothelial cells.
In one embodiment, each well of the array includes a first orifice in fluid communication with the first perfusion chamber via the first porous membrane, and a second orifice in fluid communication with the second perfusion chamber via the second porous membrane.
In one embodiment, a second layer of living cells may be disposed on a surface of the second porous membrane. In one embodiment, the second layer of living cells comprises cells selected from the group consisting of: fibroblast cells, mesenchymal cells and adipocyte cells.
In one embodiment, the central member comprises a translucent material. In one embodiment, the central member comprises an optically clear material.
In one embodiment, the first and second channel members comprise a white material. In some embodiments, the white material may allow luminescence reading of the device. For example, the top and bottom of the well containing the 3D cellular structures may be clear to allow the reading, but the chambers may be white to avoid luminescence diffusion.
In an alternative embodiment, the first and second channel members may comprise a black material. In some embodiments, the black material may allow luminescence reading of the device. For example, the top and bottom of the well containing the 3D cellular structures may be clear to allow the reading, but the chambers may be black to avoid luminescence diffusion.
Of course, the methods and systems provided herein are not constrained to any particular colors, but instead may be characterized as being reflective or absorbent with respect to wavelength ranges of the electromagnetic radiation, including in the visible range.
In one embodiment, the central member comprises polymers, preferably hard plastic materials such as polycarbonate. In one embodiment, the first and second porous membranes comprise track-etched polycarbonate. In other embodiments, the central member and channel members may comprise another injection-moldable biocompatible material. In one embodiment, the first channel member may comprise one or more alignment pins and the second channel member may comprise one or more alignment holes configured to receive the alignment pins.
In one embodiment, the first porous membrane is surrounded by a first membrane frame, and the second porous membrane is surrounded by a second membrane frame
In one embodiment a method of simulating in vivo conditions for a biological system may comprise: flowing a first fluid through a first perfusion chamber of a modular microfluidic device; passing a portion of the first fluid, via a first porous membrane, from the first perfusion chamber to a well containing one or more 3D structures of cultured biological cells; flowing a second fluid through a second perfusion chamber of the modular microfluidic device; passing a portion of the second fluid, via a second porous membrane, from the second perfusion chamber to the well; and three dimensionally growing the one or more 3D structures of cultured biological cells in the well.
In one embodiment, the method may include applying a first layer of living cells to a surface of the first porous membrane. In one embodiment, the method may include applying a second layer of living cells to a surface of the second porous membrane. In one embodiment, the first layer of living cells comprises endothelial cells. In one embodiment, the second layer of living cells comprises cells selected from the group consisting of: fibroblast cells, mesenchymal cells and adipocyte cells. The methods and systems provided herein are compatible with any number or types of components, such as cells, tissue and/or biofluids, including whole blood and constituents of whole blood, saliva, effusions, etc.
In one embodiment, the method may comprise the step of microscopically and/or spectroscopically imaging the one or more 3D structures of cultured biological cells. Microscopically imaging may include placing the device under an imaging microscope or spectroscope, and microscopically visualizing the one or more 3D structures of cultured biological cells through the device.
In one embodiment, the method may include the step of absorbance plate reading the one or more 3D structures of cultured biological cells. The absorbance plate reading may include placing the device into an absorbance plate reader, and measuring absorbance of the one or more 3D structures of cultured biological cells inside the device.
In one embodiment, the method may include the step of fluorescence plate reading the one or more 3D structures of cultured biological cells. The fluorescence plate reading may include placing the device into a fluorescence plate reader; and, measuring fluorescence of the one or more 3D structures of cultured biological cells through the device.
In one embodiment, the method may include the step of luminescence plate reading the one or more 3D structures of cultured biological cells. The luminescence plate reading may include placing the device into a luminescence plate reader, and measuring luminescence of the one or more 3D structures of cultured biological cells through the device.
In the following description, numerous specific details of the devices, device components and methods of the present invention are set forth in order to provide a thorough explanation of the precise nature of the invention. It will be apparent, however, to those of skill in the art that the invention can be practiced without these specific details.
As used herein, “biological system” is used broadly herein and refers to an in vitro biological unit of an organism that is useful in a variety of applications, including modelling and testing systems. A biological system may correspond to an organ, or a portion thereof, including cellular components thereof. For example, a biological system may be a lung, liver, intestine, heart, brain, etc., or cells thereof, including cells associated with blood vessels (e.g., endothelial cells, smooth muscle cells, and the like). A biological system may be a collection of one or more organs, tissue and/or biological fluid (e.g., blood, plasma, interstitial fluid, etc.) organized to perform one or more biological functions. For example, a biological system may be the respiratory system, digestive system, cardiovascular system, endocrine system, immune system, etc. A biological system may include a biological abnormality such as cancer. Accordingly, a “vascularized” biological system refers to a network of conduits within the biological system that is capable, at least in part, of transporting required oxygen and nutrients to the biological cells and removing waste product from the biological cell. The exchange may be similar to how exchange occurs in blood vessels, such as by diffusion. The systems are, of course, compatible with bulk media control and/or biomechanical conditions, where the cultured cells are immersed in a cell culture fluid that can be replaced or supplemented as desired.
“Three dimensional structures” or “3D structures” of cultured biological cells refers to a collection of cultured cells joined together via cell adhesion wherein the collection of cells is many cells across in all three spatial dimensions. A 3D structure of cultured biological cells may be at least 10 cells across in all three spatial dimensions. A 3D structure of cultured biological cells may be at least 100 cells across in all three spatial dimensions. Accordingly, as used herein a monolayer of cultured cells is not considered a 3D structure of cultured biological cells. In some embodiments, a 3D structure of cultured biological cells is a tissue, an organ, or a cancer mass. A 3D structure of cultured biological cells can include any eukaryotic cells that are able to self-organize in order to form a 3D structure that influences the spatial organization of the cell surface receptors engaged in interactions with surrounding cells, and may also induce physical constraints on the cells. These spatial and physical aspects in 3D cellular structures affect the signal transduction from the outside to the inside of cells, and ultimately influence gene expression and cellular behavior in a manner closer to that of the in vivo environment than a standard cellular monolayer culture. The systems and methods provided herein are compatible with a range of biological cell sources, including cells that have not been cultured, so long as the seeded cells are capable of subsequent cell culture after being introduced to the microfluidic device.
The devices disclosed herein provide a platform technology mimicking human ovarian TME, including culture of 3D cancer structures in close contact with several tissue compartments and extracellular matrix, in dynamic flow condition, while having high-throughput capacity and a ready-to-use configuration for subsequent biological analysis. Indeed, the device is highly customizable for any type of cells. For example, the different chambers may be controlled independently allowing cell culture in different medium, flow condition (shear stress, flow rate, etc.), gas environment, etc. Moreover, the device may be directly compatible with most of the common experimental methods and thus ready-to-use for microscopy imaging or plate reader analysis.
In one embodiment, the tumor-on-chip devices disclosed herein combine the advantages of standard cell culture (human cells, easy to handle, low cost) and animals models (complex spatial organization, interaction of cells) in order to mimic in vitro the human in vivo TME. This approach is especially beneficial for the study of cancer with a specific TME, such as ovarian cancer of which TME includes not only vascular and stroma system but also the unique presence of peritoneal cavity which drives many factors responsible of toxicity, metastasis dissemination, etc.
In one example, the different perfused chambers provide functionality to mimic and control independently both the flow condition of vascular system and peritoneal cavity. In some embodiments, the device allows for flexible design workflow (numbers of replicates, conditions, high-throughput, etc.) and analysis with diverse bottom/top optical readers.
In one embodiment, a tumor-on-chip may grow ovarian cancer cells spheroids, surrounded by functional monolayer of endothelial cells and fibroblasts cells seeded on membranes and cultured in representative dynamic condition. The device is user friendly by being directly compatible with microscopes and microplate readers and allowing investigation of several biological samples at the same time. If desired, fasteners such as clips may be used to ensure a tight fluid seal while facilitating convenience as to accessing the different cell compartments. The devices and methods are compatible with a range of membranes, including membranes with different pores, such as different pore density and/or sizes. Any type of cell can be used with the membranes, so long as the cell is capable of adhering to the membrane and is compatible with the instant cell culture techniques.
The high number of anti-cancer drug candidates that fail to advance past the preclinical research stage is due, at least in part, to the lack of efficient in vitro models that can recapitulate in vivo human tumor microenvironment (TME) including 3D structure, multi-cellular components and dynamic flow environment. That is particularly true for some cancers, such as ovarian cancer, with a complex and specific molecular and cellular TME. The interaction of the TME with cancer cells plays a pivotal role in tumor development, progression and drug resistance (Worzfeld et al., 2017). Through the convergence of tissue engineering and microfluidics, small devices known as organs-on-a-chip have been engineered with the potential to improve the knowledge of such complex environments (Zhang et al., 2018).
Recently, cancer-on-a-chip models have emerged as a tool to study the TME. They contain small chambers for cell culture, enabling control over local gradients, fluid flow, tissue mechanics, and composition of the local environment (Tsai et al., 2017). Prior art cancer-on-a-chip systems still have several limitations. First, most of them implement co-culture using cancer cells and only one other cell type, thus neglecting other compartments such as vascular tissue, connective tissue or immune system. Second, the cancer-on-a-chip systems of the prior art don't provide for a three dimensional cell growth and can't culture spheroids/organoids in contact with other cell types. Third, these prior art cancer-on-a-chip systems are complex and arduous to set up and work with, and thus do not allow high-throughput analysis and/or are not directly compatible with common analytical methods. Hence, there is a need to develop a new platform enable simulation of the human TME, recapitulating the 3D structure of tumors and the main cellular compartments while allowing easy access for subsequent required biological analysis.
Provided herein are in vitro platforms that can mimic in vivo human organ microenvironment via modular microfluidic devices for simulating physiological conditions of biological systems, in order to provide new models for biomedical research. The microfluidic devices disclosed herein may be used to assess the efficacy of new natural compounds (e.g. withanolides) as cytotoxic agents for ovarian cancer. It is believed that 3D structure and multiple cellular components of TME widely influence ovarian cancer response to drug treatment. This is demonstrated herein via data regarding the difference of the efficacy of anti-proliferative drugs as applied to monolayer (2D) cellular structures as compared with spheroids/organoids or organ-on-chip cell culture. The tumor-on-chip microfluidic devices disclosed herein provide a new approach to assess cytotoxic activity of anticancer drugs on both cancer and normal cells and thus recapitulate the real in vivo effect of the drug in the ovarian TME.
In one embodiment of the present invention, the microfluidic device allows separation of interconnected chambers for culturing, in dynamic mode, three different tissue compartments of the TME: cancer spheroids (epithelial cancer cells), vascular tissue (endothelial cells) and connective tissue (fibroblasts).
Without wishing to be bound by theory, it is believed that the half-maximal inhibitory concentration (IC50) of withanolides is a function of the three dimensional nature of tumor structures and cell interactions therein. The tumor-on-chip microfluidic devices disclosed herein mimic the ovarian TME and are suitable for assessing cytotoxic effects of anti-cancer drugs in a simulated in vivo condition. Thus, the devices herein shed light on important questions in ovarian cancer such as immunotherapy resistance/toxicity or the role of peritoneal fluid in metastasis dissemination.
As shown in
The modular microfluidic devices 51 may be configured to allow direct analysis in a plate reader. For example, the distance between each well in the strip may be configured to be compatible with the spacing of a 96-well plate. Furthermore, the microfluidic devices may be comprised of a clear or translucent material. Thus, as shown in
The device 51 may be configured for easy opening and to provide access to cells seeded on the membranes. Plastic material may be configured to have improved optical transparency to improve microscopic observation. The devices are useful for the simultaneous culture of multiple different cell types with different geometries, including 3-D dimensional configurations in the well (e.g., spheroids associated with certain tumors) and monolayers (e.g., endothelial cells associated with blood vessels and fibroblasts or mesothelial cells associated with the peritoneal fluid)
The withanolides withaferin A (WFA) and withanolide D (WD) were investigated for cytotoxicity when used to treat SKOV3 ovarian cancer cells. As shown in
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The device may be made with multiple materials, requiring several steps. The outer manifolds may be 3D printed using a Form2 SLA printer, creating the chambers 205, 206 and barbs 221 along with alignment pins 231 for assembly. The center member 101 may be machined Polycarbonate. The first and second channel members 201, 202 may be pressed together with double-sided medical PSA. Track-etched Polycarbonate porous membranes (8 μm pores) separate the chambers. Once the device 51 is assembled, it is cleaned with 70% ethanol and allowed to dry. Then, it is irradiated with X-Rays at 10 Gy (3 Gy/min) and kept in sterile condition until cell culture.
Cell Culture
Before proceeding to cell seeding, porous membranes were coated with collagen (50 μg/mL) for 4 hours. Endothelial HUVEC cells (ATCC® CRL-1730™) were first seeded on membrane into one of the chambers and allowed to attach for 2 hours. The device was then flipped 180° and Human Ovarian Fibroblast (HOF, Cat. #7330, ScienCell Research Laboratories) were seeded on the membrane of the other chamber and allowed to attach for 2 hours. After cell seeding, each chamber was perfused with respective medium (F-12K Medium (ATCC 30-2004) supplemented with 0.1 mg/mL heparin, 5 mL endothelial cell growth supplement (ECGS; BD Biosciences catalog #354006) and 10% of Fetal bovine serum (FBS) for HUVEC cells (ATCC® CRL-1730™) and Fibroblast Medium (FM, Cat. #2301, ScienCell Research Laboratories) for HOF cells) at a flow-rate of 10 μL/min for 7 days. In parallel SKOV3 cells (ATCC® HTB-77™) were maintained in McCoy's 5A medium supplemented with 10% of Fetal bovine serum (FBS) and 1% of antibiotics mixture streptomycin/penicillin at 37° C. in 5% CO2 atmosphere. The SKOV3 cells were seeded into a 96 round-bottom well ultra-low affinity plate at concentration of 10,000 cells/well and allowed to grow for 5 days. After 5 days, the resulting SKOV3 spheroids were transferred into the wells of the tumor-on-chip device. After the SKOV3 spheroids were loaded and HUVEC and HOF cells were seeded for a period of 7 days, the medium flowing through the HUVEC chamber was changed and replaced by medium-containing peripheral blood mononuclear cells (PBMCs). The PBMCs were previously isolated by using Ficoll gradient density medium from whole blood (healthy individual, BioChemed). PBMCs were perfused in the device for 1 day.
Cellular and Molecular Analysis
MTT assay was performed directly on spheroids, over a period of 5 days, to assess whether they were able to grow into the device. To assess cell communication and migration of PBMCs into the spheroids chambers, chemoattractant SDF-1a was injected into spheroids chambers and PBMCs were stained 24 hours later with CD2 antibody to detect, specifically, T-cells and NK cells, while the SKOV3 spheroids were stained with pan-cytokeratin (Abcam, ab7753). Cytokine profile was analyzed to investigate if secretion was modified by the presence of spheroids and/or fibroblasts. The device was disassembled at 2 and 7 days after initial HUVEC and HOF seeding. The porous membrane was removed to be analyzed using fluorescence analysis. Each porous membrane was cut into several pieces and was stained with α-tubulin (Santa Cruz, sc-5286), Pericentrin (abcam, ab4448), Connexin (Cell Signaling, P17302), Vimentin (abcam, ab92657), Ki67 (abcam, ab15580), β-catenin (Santa Cruz, sc-7963), α-catenin (abcam, ab51032), Ecadherin (ThermoFischer, 13-1700), VE-cadherin (Santa Cruz, sc-9989), Integrin β1 (Santa Cruz, sc-13590) and Phalloidins (Invitrogen, MAN0001777) antibodies to assess cell shape, cell junctions and cell proliferation. A full membrane was also stained with a live/dead cell viability assay to assess cell viability and distribution over the membrane.
Cytotoxicity Activity of Withanolides Compounds on Ovarian Tumor Microenvironment by Using Tumor-On-Chip.
The specifics of the TME may affect the cancer cells response to drug treatment. In this regard the effect of withanolides on SKOV3 speroids was investigated. It is believed that IC50 of withanolides is different when drugs are delivered to SKOV3 spheroids cultured in standard cell culture versus spheroids incubated in a tumor-on-chip device of the present disclosure. Thus, SKOV3 cells, cultured as: (1) a monolayer and (2) as spheroids in standard 96-well plates without contact with any other type of cells or on the tumor-on-chip, were exposed to different concentrations of WFA, WD and analogues as well as carboplatin/paclitaxel cocktail.
Experimental Protocol:
SKOV3 cells were maintained in the same condition as described above. Cells were seeded as: (1) a monolayer in 96-flat bottom well plates and as (2) spheroids in 96-round bottom well ultra-low affinity plates at a concentration of 5000 cells/well. The next day after cell seeding, the cells cultured as monolayer were exposed to a range of concentrations (0.1 to 80 μM) of WFA, WD, 15β-hydroxyWFA and 15β-hydroxyWD and a range of concentrations (20 to 320 μM and 3 to 50 μM) of carboplatin and paclitaxel, respectively, for 3 days. The cells cultured as spheroids were allowed to grow for 5 days before being exposed to the same drug concentrations for 3 days. Half of the spheroids were transferred into the tumor-on-chip before being exposed to the drugs following the same protocol previously described. The tumor-on-chip devices were previously seeded with HUVEC cells, HOF and PBMCs according to the same protocol described previously. After 3 days, the cells were exposed to MTT assay and absorbance was directly read from the plate and the tumor-on-chip on a BioTek® Epoch™ Microplate Spectrophotometer after overnight incubation at 37° C. For SKOV3 spheroids, another experiment was performed to measure growth rate over a long period of time. Spheroids were exposed to a unique dose of drug (to be determined according to IC50 curves) for 3 days and spheroids are then measured 2 or 3 times a week with a Nikon TS100-F microscope over a period of >20 days.
Statistical Analysis:
The estimation of IC50 values was performed in GraphPad Prism version 7.00 for Windows (GraphPad Software Inc., La Jolla, Calif.) using dose response curve fitting ((inhibition) vs normalized response (variable slope)) of SKOV3 cells treated with different doses of drugs. A Mann-Whitney test or Student's t-test was then used to compare IC50 of the different drugs according to the respective culture condition. p-value<0.05 was considered statistically significant. Each experiment was independently repeated 3 times with at least 4 internal replicates each time and expressed as a mean±standard error (SEM).
The 3D biological tumor cells on chip model was validated by showing the difference in cytotoxicity (as determined by IC50 values) of drugs exposed to SKOV3 cells cultured as (1) a monolayer and (2) unique spheroids or as spheroids into a co-culture device perfused with immune cells. This demonstrates that biological cells in the instant device are alive, responsive to internal and external stimuli. Furthermore, the devices and methods can facilitate testing of promising compounds, such as withanolides, for new anticancer drugs for ovarian cancer.
The devices and methods provided herein are compatible with a range of biological systems, thereby facilitating a range of modeling and testing applications, including non-cancer soft tissue test, such as drug efficacy, clearance and/or toxicity.
For example, mixed populations of mammalian and bacterial cells are compatible, with one compartment having mammalian cells and the other bacterial cells, thereby facilitating modeling of, and impact by, the microbiome.
Despite the unheralded success of immune checkpoint blockade in delivering durable responses for some patients with non-small-cell lung cancer (NSCLC), the majority of patients do not respond to anti-programmed cell death protein 1 (PD-1) immune checkpoint inhibitors (ICIs) [1]. Yet, radiotherapy (RT) can increase the immunogenicity of cancer cells and can improve long-term antitumor responses when combined with ICIs [2]. However, there are still unknown practical considerations that may impact the efficacy of RT combined with immunotherapy, including the dose, fractionation and scheduling of RT with ICIs. In particular, stereotactic ablative RT (SABR) is a technological advancement that delivers higher doses of RT, using a hypofractionated schedule and with greater conformity. Thus, SABR reduces normal tissue toxicity and improves control of the primary tumor [3]. Nonetheless, evidence is required to elucidate whether SABR in combination with ICIs has superior immune-priming capability to conventional RT and study the optimal radiation regimen. The current lack of reliable data to investigate such mechanisms is mainly due to the inaccuracy of pre-clinical models that cannot reproduce the complexity of human tumor microenvironment (TME). Standard human in vitro models are not able to recapitulate the complete interaction of tumor-stroma-immune system and animal in vivo models suffer of important physiological differences. Hence, there is a critical need to exploit new bioengineered systems to accurately reproduce human TME and investigate its response to ionizing radiation in combination with immunotherapy.
The optimal SABR radiation regimen may be determined via the modular microfluidic devices disclosed herein. In one embodiment, the modular microfluidic devices maximize the lung tumor immune response while minimizing damages to normal tissue in combination with an anti-PD1 therapy by using a bioengineered platform recapitulating accurately human lung TME. The dynamic “tumor-on-chip” platform disclosed herein recapitulates the interaction of three-dimensional (3D) tumor structures with vascular compartment and the connective tissue. Published studies suggest that SABR may be more immunogenic than conventional RT [4,5].
In one embodiment, the modular microfluidic devices of the present disclosure allow determination of the optimal SABR radiation regimen to maximize NSCLC tumor response to anti-PD1 immunotherapy. Without wishing to be bound by theory, it is believed that a specific SABR regimen, to be determined as high single dose or hypofractionation, enhances tumor immunogenic cell death (ICD) and proimmunogenic inflammatory balance promoting anti-PD1 efficiency.
Furthermore, and it is believed that that the SABR regimen combined with anti-PD1 therapy may prevent damage to surrounding normal connective tissue compared to conventional RT.
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Therefore, the device provides the appropriate platform to investigate the critical need of better understanding the effect of RT, and in particular SABR radiation regimen, on the lung TME immune response, when combined with immunotherapy such as anti-PD1 agent. The organ-on-chip technology may be combined with 3D cell culture to identification of the optimal SABR regimen to maximize the lung TME immune response to anti-PD-1 immunotherapy while minimizing damages to normal tissue
One embodiment of the modular microfluidic device is shown in
Device 50 may include a porous membrane 350 supported and surrounded by a membrane frame 300. The membrane frame 350 may be sandwiched securely in place between the channel member 200 and the web portion 160 of the central member 100. The channel member 200 may include a depression 262 (best shown in
Additionally, the web portion 160 may include well access ports 190 formed therein, providing access to the ports 170 from above. A bottom edge 194 of the access port 190 terminates in the well. A top edge 192 of the access port terminates on the upper surface of the flange portion 150.
The first channel member 200 includes barbed connections 220 at each end to facilitate the flow of perfusion fluid. The barbed connections may include flow distributors 210. The flow distributors 210 may terminate inside the perfusion chamber in a distributor throat 260. The illustrated distributor throat 260 is hemispherical. Of course, in other the embodiments the distributor throat may be another shape. The central member 100 may include alignment pins 130 on the web portion 160 in order to align and secure the first channel member 200 in place. In this regard, the first channel member 200 may include alignment holes 230 configured to snugly receive the alignment pins 130.
The modular microfluidic device 50 may be assembled and functionalized via a multi-step process. First, the channel member 200 may be 3D printed using a Form2 SLA printer, creating the large chamber and barbs 220 along with alignment pins 130 for assembly. In parallel, the central member 100 may be machined in acrylic material. Channel member 200 may be then sealed to central member 100 with double-sided medical PSA. Track-etched polycarbonate porous membranes 350 (8 μm pores) may separate the channels from the wells 170. Once the device is assembled, it may be cleaned with 70% ethanol and let dry. Then, it may be irradiated with X-Rays (>500 Gy) and kept in sterile condition until use.
Once assembled and before cell seeding, porous membranes may be functionalized with Collagen type I (50 μg/mL). After one-hour incubation, Human Lung Microvascular Endothelial Cells (HLMVECs) may be then seeded in one chamber. After cell attachment (˜1 h), the device may be flipped and normal lung fibroblasts (IMR-90) may be seeded in the other channel member. HLMVECs and IMR-90 may be cultured for 24 h in static condition until flow is initiated for 5 days. Flow rate in the vascular chamber may be 300 μL/min to generate a shear stress of 1.7 dyne/cm2, as usually encountered in tumor blood vessels [38]. Flow rate in the fibroblasts chamber may be 20 μL/min to reproduce cell surface shear stress (0.1 dyne/cm2) generated by interstitial flow within the TME [39].
In parallel, lung cancer cells (A549) may be grown in 96-well plates round bottom ultra-low attachment for 5 days to form spheroids. Initial numbers of cells may be adapted for each cell lines in order to obtain after 5 days spheroids with a size comprised between 0.5 and 1 mm. After 5 days, spheroids may be embedded in Matrigel (50 μL), loaded into the device and allowed to adapt for 24 hours. After spheroids loading, peripheral blood mononuclear cells (PBMCs) may be isolated by density gradient centrifugation from whole blood. PBMCs may be then resuspended in HLMVECs medium (2.106 cells/mL) and injected in the vascular compartment for 24 h before irradiation. After PBMCs injection, flow may be adjusted to 10 μL/min to match blood velocity in tumor blood vessels and favor extravasation and PBMCs infiltration.
Irradiation may be performed with a TrueBeam™ linear accelerator (Varian, Palo Alto, Calif.) utilizing a 6 MV energy, 600 MU/min dose rate, and the 120 Millennium multileaf collimator (MLC). An assembled ASTEROIDS unit, filled with a medium with similar density characteristics as the seeded cancer lines as described above, may be scanned using a Philips Brilliance CT Big Bore with a technique, scan length, and field of view appropriate to the device dimensions. A slice thickness of 1 mm may be utilized. The DICOM image dataset may be imported into the Varian Eclipse clinical treatment planning system. Treatment plans may be designed to deliver known amounts of radiation dose (see next following paragraph) to the target volumes using the AcurosXB 15.6.06 dose calculation algorithm and a 1 mm dose grid. Mobius3D software, which utilizes an independent beam model and dose calculation algorithm, may be used to perform an independent dose calculation to verify the accuracy of the treatment planning system dosimetry. The clinical daily quality assurance routine may be performed using a Sun Nuclear Daily QA™ 3 device (dose output verification) and Varian's integrated machine performance check (imaging, MLC, mechanical, and dose output verifications). The seeded device may be positioned on the TrueBeam linear accelerator's 6 degree of freedom treatment table. Onboard kV and MV imaging may allow for pre-treatment visualization. Small translational and rotational movements may be made by the table for accurate alignment of the device to within 1 mm. The designed radiation treatment plans may then be delivered.
The device may be irradiated at single dose 0, 8 and 24 Gy at a dose rate of 600 cGy per minute. To assess the effect of fractionation, 3×8 Gy (8 Gy/day for 3 continuous days, 24 Gy total dose) regimen may also be performed. For each data point, biological outcomes may also be measured in presence of anti-PD-1 antibody. To determine the most appropriate protocol for drug delivery, anti-PD-1 compound may be tested on A549 spheroids seeded in 96 well plates exposed to different anti-PD-1 concentration (0.001 to 10 μg/mL). The antibody may be delivered either 1 or 24 h before irradiation and injection may be pursued after irradiation at different intervals. Spheroids growth may be assessed by measuring spheroid size directly within the device every two days for 2 weeks under microscope. Volume of spheroids may be calculated using the formula 4/3π(d1+d2/4)3 where d1 is the length diameter and d2 the width diameter of the spheroid. The concentration and time of delivery may be chosen for the protocol that showed the minimal spheroids size after 2 weeks. Biological endpoints may be measured and analyzed at 2- and 7-days post-irradiation.
For each data points, three devices will be irradiated with 6 loaded spheroids in each of them. This sample size is determined for a two Independent sample study design with continuous endpoint by the equation n1=(σ12+σ22/K)(z1-α/2+z1-β)2/|μ2−μ1|2 with an enrollment ratio K=n1/n2=1, assuming a probability of type-I error (a) of 0.05 and a risk of type-II error (β) of 0.2. σ1 and σ2 are the variance of anticipated mean μ1 and μ2 (extracted from published studies) and z is the critical Z value for a given α or β. A total of 48 devices may be irradiated for this aim (4 radiation regimen×2 drug concentration (with and without anti-PD-1)×2 timing points×3 replicates).
Analytic Methods
In order to estimate initiation of ICD, ATP and HMGB1 release and CRT membrane exposure may be assessed. ATP may be measured both in the supernatant and in the spheroids. Supernatant may be collected after irradiation from vascular compartment and connective tissue. Spheroids may be unloaded and analyzed in a 96 well plates. ATP assay may be performed using colorimetric ATP Detection Assay Kit (Abcam). Concentration of ATPsamples may be calculated from standard curve values and increase of ATP release may be assumed whether the ratio [ATPextracellular]/[ATPintracellular] of irradiated samples is higher than the ratio of sham-irradiated samples. In order to estimate the extracellular release of HMGB1, ELISA may be performed on supernatant by using the gold-standard HMGB1 ELISA Kit (Tecan). Release of HMGB1 may be assessed by comparing supernatant from irradiated samples to sham-irradiated sample in presence or not of anti-PD-1. Positive and negative controls available in the kit may be used to create standard curves and quantify released [HMGB1]. Finally, the presence of membrane CRT may be assessed by immunofluorescence microscopy (IF). Spheroids may be fixed with 0.25% paraformaldehyde but no permeabilized and incubated with primary anti-CRT antibody. After washing and incubation with fluorescence-labeled secondary antibody, fluorescence may be quantified by confocal microscopy. Exposure to secondary antibody alone may be used as a negative control.
To assess significance when comparing two groups, the Bartlett test may be first used to test if the tested biomarkers present significant homogeneous or heterogeneous variances between groups. Then, significance of two-group comparisons may be calculated using the Mann-Whitney nonparametric test (for heterogenous variances) or 2-tailed Student's t-test (homogeneous). P values of <0.05 may be considered significant.
In order to determine which radiation regimen may generate a proimmunogenic signature favorizing anti-PD-1 response, molecular signals on HLMVECs and A549 spheroids as well as secreted molecules I supernatant may be assessed. First, VCAM-1 may be detected by IF on HLMVECs attached on membrane. Second, NKG2DL and ICAM-1 expression may be assessed by IF on spheroids. Fluorescence signal intensity may be compared between membranes/spheroids exposed to different regimen by either nonparametric Mann-Whitney test or 2-tailed Student's t-test. Finally, TNF-α, IFN-α/β, IFN-γ, IL-1β, CXCL9, CXCL10 and CXCL16 may be simultaneously detected using Human Magnetic Luminex Assay (R & D systems) while TGF-β will be quantified by Human TGF-beta 1 Quantikine ELISA Kit (R & D systems) from collected supernatants. Cell culture media alone may be used as negative control. Cytokine values may be log-transformed and data may be first probed for normality using the Bartlett test.
Depending on result, either the nonparametric Mann-Whitney test or 2-tailed Student's t-test may be used to compare the individual cytokine level between two groups. A one-way ANOVA with Tukey's multiple comparison may be used to compare the effect between sham-, single and fractionated irradiated groups in presence or not of anti-PD-1 antibody. In order to assess which radiation regimen provides the best pro-immunogenic response, the combined cytokine performance may be calculated based on SVM analysis or receiver operating characteristic (ROC) curves. The generalized ROC criterion may find the best linear combination of cytokines such that the area under the ROC curve (AUC) is maximized. Viability of immune cells, fibroblasts, cancer cells and HLMVECs may be assessed by Live/Dead assays.
In addition to cytokine profile, PBMCs population may also be investigated. Indeed, TReg cells and tumor-associated macrophages have been associated with pro-tumor functions, whereas CD8+ T cells have been associated with anti-tumor functions[40-42]. Therefore, flow cytometry analysis may be performed on supernatant from spheroids chambers and connective tissue to assess immune cell infiltration depending on radiation regimen by using CD45+/CD25+/FOXP3+ staining (TReg cells), CD8+/CD4−/CD45+ staining (CD8+ T cells), CD45+/CD68+/CD80−/CD163+ staining (M2-macrophages). As negative control, samples incubated with secondary antibodies only may be used. Percentage of TReg cells, CD8+ t cells and M2 macrophages may be calculated according to the total number of infiltrating cells in a sample and compared between the different radiation regimen by either nonparametric Mann-Whitney test or 2-tailed Student's t-test.
Finally, spheroids growth will be monitored every day during the experiment duration by measuring spheroids size as described above in order to compare anti-PD-1 treatment efficiency in combination with the different radiation regimens.
To assess any toxicity from the different treatments, direct effect on normal fibroblasts may be assessed. First, radiation-induced DNA damages may be assessed by γH2AX/53BP1 foci assay. IMR90 may be collected at 2 and 7 days and stained for IF. Co-localized γH2AX/53BP1 foci from at least 100 cells from three independent experiments may be manually counted. Ratio of radiation-induced γH2AX/53BP1 foci (with the different radiation regimen) to sham-irradiated in presence or not of anti-PD-1 as well as absolute number of γH2AX/53BP1 foci for each condition may be calculated. Differences with control (sham-irradiated/sham-anti-PD-1 exposed) may be determined with Student t-test.
Second, IMR90 phenotype changes may be assessed to determine if the fibroblasts turn into a pro-inflammatory/pro-fibrotic phenotype [49] under certain radiation regimens. Thus, a series of experiments may be performed to monitor whether fibroblasts have been activated by any of the treatments. Gene expression assay of extracellular matrix & adhesion molecules (Qiagen, RT2 Profiler™ PCR Array) may be performed to identify any potential changes in fibroblast secretory function, including overexpression of collagen and matrix metalloproteinase genes. Half of the membrane may be used to lyze directly the fibroblasts and mRNA will be extracted using RNA extraction kit (Qiagen) then qualitatively assessed with Bioanalyzer (Agilent). qPCR may be performed according to manufacturer protocol. Ct values may be normalized with five different housekeeping genes and analyzed according to delta Ct method [50] by comparing samples to sham-irradiated/sham-anti-PD-1 exposed sample. Gene expression may be significantly modified if the associated p-values are less than 0.05 and fold changes (FC) is greater than 1.5 or less than 0.66. In addition, collagen secretion may be assessed by total collagen assay (Abcam) in supernatant. Since activated fibroblasts also demonstrate cytoskeletal remodeling [51], vimentin and a-smooth muscle actin staining may be performed by IF as well as other activation biomarkers such as octamer-binding transcription factor-4 (Oct4), Nanog, collagen I, sex-determining region Y-box 2 (Sox2), chemokine receptor-4 (CXCR4), fibronectin. Proliferation biomarkers, including Ki67, may also be assessed. Finally, IMR90 senescence may be assessed by β-galactosidase assay (Cell Signaling). Membrane seeded IMR90 may be cut at the end of the experiment and put in IMR90 medium for 24 hours to perform in vitro assay. Senescence may be visualized by blue staining under bright field microscope. At least 100 cells (from 3 independent experiments) may be counted and t-test assay may be performed to determine if differences between samples and sham-irradiated/sham-anti-PD-1 exposed sample are significant. Negative control may be the sham-irradiated and sham-drug exposed device membrane. For positive control, IMR90 seeded on membrane may be exposed to etoposide (12.5 μM) for 24 hours and incubated 3 days prior imaging.
References Associated with Example 2
Curr Opin Immunol. 2014; 27: 1-7. doi:10.1016/j.coi.2013.12.005
All references throughout this application, for example patent documents including issued or granted patents or equivalents; patent application publications; and non-patent literature documents or other source material; are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the disclosure in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference).
The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. The specific embodiments provided herein are examples of useful embodiments of the present invention and it will be apparent to one skilled in the art that the present invention may be carried out using a large number of variations of the devices, device components, methods steps set forth in the present description. As will be obvious to one of skill in the art, methods and devices useful for the present methods can include a large number of optional composition and processing elements and steps.
When a group of substituents is disclosed herein, it is understood that all individual members of that group and all subgroups, are disclosed separately. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure.
Every formulation or combination of components described or exemplified herein can be used to practice the invention, unless otherwise stated.
Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the claims herein.
All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. References cited herein are incorporated by reference herein in their entirety to indicate the state of the art as of their publication or filing date and it is intended that this information can be employed herein, if needed, to exclude specific embodiments that are in the prior art. For example, when composition of matter are claimed, it should be understood that compounds known and available in the art prior to Applicant's invention, including compounds for which an enabling disclosure is provided in the references cited herein, are not intended to be included in the composition of matter claims herein.
As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.
One of ordinary skill in the art will appreciate that starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this invention. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
This application claims the benefit of priority to U.S. Provisional Patent Application No. 62/868,572 filed Jun. 28, 2019, which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/039944 | 6/26/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62868572 | Jun 2019 | US |
