Method, system, and device for detecting drug resistance and cell death mechanism in cancer

Abstract

The invention provides a system and method for assaying the geometric diameter and electrical diameter of a cell. The method may include steps of determining the susceptibility of the cell to a dug. The invention further comprises a method of treating cancer comprising identifying a population of cancer cells from a subject as susceptible to a drug and administering that drug to the subject.

Description

Attorney Docket No. 206085-0196-00WOMETHOD, SYSTEM, AND DEVICE FOR DETECTING DRUG RESISTANCE AND CELL DEATH MECHANISM IN CANCERCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 737,986, filed on December 23, 2024, which is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION

[0002] Chemotherapy has been the mainstay therapy for several different cancers with proven success; however, its effectiveness is often limited by the tumor heterogeneity and the development of drug resistance. In contrast, targeted therapies directed towards the drivers of carcinogenesis are more effective and have fewer side effects, as they are designed to interfere directly with the specific molecular mechanisms responsible for tumor development and progression while keeping normal cells unaffected (Katsios et al., 2013, Biomarkers in Medicine.7:79-82). Consequently, patients often experience better outcomes, less toxicity, and an improved quality of life. Unfortunately, patients eligible for targeted therapies represent only a small fraction of the affected population (Flaherty et al., 2020, Journal of Clinical Oncology. 38:3883-3894). Moreover, even among personalized therapies that reach the clinical setting, (Batis et al., 2021, Advance Drug Delivery Reviews. 176:113854) only a small subset of patients respond effectively (Marquart et al., 2018, JAMA Oncology. 4:1093-1098; Haslam et al., 2021, Annals of Oncology. 32:926-932) while many of those who respond to the treatment are likely to develop drug resistance after initial remission (Flaherty et al., 2020, Journal of Clinical Oncology. 38:3883-3894).

[0003] To address these challenges, precision oncology uses a multitude of tools to predict a patient's response to therapy, including DNA and RNA sequencing, protein expression profiling, and multiomics-based biomarker detection. However, there are certain challenges involved in these approaches. For example, while the identification of single genes has yielded promising clinical results, a more in-depth view of clinical cases often reveals complex networks of interacting gene expressions that ultimately affect cancer development and metastasis. On the other hand, research into high-throughput multi-omics has yielded enormous volumes of dataAttorney Docket No. 206085-0196-00WOwhich are not easy to translate into disease phenotypes clinically (Katsios et al., 2013, Biomarkers in Medicine. 7:79-82; Dumbrava et al., 2018, Expert Opinion on Drug Discovery.13:685-690).

[0004] Therefore, there remains an unmet need to create rapid and effective strategies to identify the emergence of drug resistance early on in tumors having high heterogeneity. The present invention meets this unmet need.SUMMARY OF THE INVENTION

[0005] In some aspects, the invention provides a system for measuring the geometric diameter and electrical diameter of a cell, comprising: a planar body with a microchannel comprising an inlet and an outlet, wherein the microchannel comprises a centrally located constriction; a split-ring resonator (SRR) positioned on the body comprising an inner ring and an outer ring, wherein the inner ring comprises a gap in the ring; and a Coulter counter positioned on the body comprising first and second electrodes, wherein the constriction of the microchannel, the gap of the inner ring, and the first and second electrodes of the Coulter counter are aligned to form a sensing region for measuring a sample comprising at least one cell.

[0006] In some embodiments, the inner ring comprises a first end comprising a tabbed portion, and a second end comprising a slotted portion, wherein the tabbed portion at least partially extends into the slotted portion to form the gap.

[0007] In some embodiments, the tabbed portion is asymmetrically positioned in the slotted portion.

[0008] In some embodiments, the width of the inner ring tapers from the second end to the first end.

[0009] In some embodiments, the width of the inner ring is less than the width of the outer ring.

[0010] In some embodiments, the first and second electrodes of the Coulter counter are separated by a distance along the length of the body ranging between 10 pm and 1000 pm.Attorney Docket No. 206085-0196-00WO

[0011] In some embodiments, the system further comprises a signal generator and a first lock-in amplifier electrically connected to the outer ring of the SRR, and a second lock-in amplifier electrically connected to the electrodes of the Coulter counter.

[0012] In some embodiments, the SRR is a high-frequency sensor and the Coulter counter is a low-frequency sensor.

[0013] In some embodiments, the sample comprising at least one cell is passed from the inlet to the outlet across the sensing region to measure the geometric diameter and electrical diameter of the at least one cell.

[0014] In some aspects, the invention relates to a method of measuring the geometric diameter and electrical diameter of a sample comprising at least one cell using a system for measuring the geometric diameter and electrical diameter of a cell, comprising: a planar body with a microchannel comprising an inlet and an outlet, wherein the microchannel comprises a centrally located constriction; a split-ring resonator (SRR) positioned on the body comprising an inner ring and an outer ring, wherein the inner ring comprises a gap in the ring; and a Coulter counter positioned on the body comprising first and second electrodes, wherein the constriction of the microchannel, the gap of the inner ring, and the first and second electrodes of the Coulter counter are aligned to form a sensing region for measuring a sample comprising at least one cell.

[0015] In some aspects, the invention relates to a method of identifying a population of cells as susceptible or resistant to a drug comprising measuring the geometric diameter and electrical diameter of the at least one cell.

[0016] In some embodiments, the method comprises exposing the at least one cell to the given dose of the drug and subsequently measuring the geometric diameter and electrical diameter of the at least one cell.

[0017] In some embodiments, the method comprises: a) providing a system comprising: a planar body with a microchannel comprising an inlet and an outlet, wherein the microchannel comprises a centrally located constriction; a split-ring resonator (SRR) positioned on the body comprising an inner ring and an outer ring, wherein the inner ring comprises a gap in the ring; and a Coulter counter positioned on the body comprising first and second electrodes, wherein theAttorney Docket No. 206085-0196-00WOconstriction of the microchannel, the gap of the inner ring, and the first and second electrodes of the Coulter counter are aligned to form a sensing region; and b) contacting the at least one cell to the sensing region.

[0018] In some embodiments, the method comprises applying a high-frequency signal to the SRR and obtaining the change in capacitance at the gap in the inner ring to measure the electrical diameter of the at least one cell, and applying a low-frequency signal to the Coulter counter and obtaining the change in current across the electrodes to measure the geometric diameter of the at least one cell.

[0019] In some embodiments, the method comprises comparing the geometric diameter and electrical diameter of the at least one cell exposed to the drug with a baseline measurement.

[0020] In some embodiments, the baseline measurement is the geometric diameter and electrical diameter of the at least one cell without being exposed to the drug.

[0021] In some embodiments, the cell is determined to be susceptible to the given dose of the drug when the geometric diameter is increased or decreased by at least 5%.

[0022] In some embodiments, the cell is determined to be susceptible to the given dose of the drug when the electrical diameter is increased or decreased by at least 5%.

[0023] In some embodiments, the cell is a cancer cell. In some embodiments, the cell is a tumor cell.

[0024] In some embodiments, the drug is a cytostatic, cytotoxic, or mixed-effect drug.

[0025] In some aspects, the invention provides a method of treating cancer in a subject comprising determining that a population of cancer cells from the subject is susceptible to a drug by a method of identifying a population of cells as susceptible or resistant to a given dose of a drug comprising measuring the geometric diameter and electrical diameter of the at least one cell, and administering the drug to the subject.Attorney Docket No. 206085-0196-00WOBRIEF DESCRIPTION OF THE DRAWINGS

[0026] The foregoing purposes and features, as well as other purposes and features, will become apparent with reference to the description and accompanying figures below, which are included to provide an understanding of the invention and constitute a part of the specification, in which like numerals represent like elements, and in which:

[0027] Figure 1, comprising Figures 1 A-1D, depicts a schematic representation of the sensor operating principles. Figure 1 A shows how the increase in the frequency of the alternating electric field ends up penetrating into the cell interior as it no longer suffers from chargeshielding effects of the ions, since ion movement cannot keep up with the speed of the alternating electric field at GHz frequencies. Figure IB shows the workflow followed to generate and test resistant cancer cell line from their parental counterparts. Figure 1C shows the workflow followed to generate patient derived organoids and measure their susceptibility to Gefitinib (Gef) and Bortezomib (Brz) drugs (dark arrows). Light arrow indicates an alternative, and faster path for testing single cells. Figure ID shows how parental cells were differentiated from their resistant variants by looking at the change in the measured permittivity when exposed to the drug.

[0028] Figure 2, comprising Figures 2A and 2B, depicts the results of example experiments demonstrating the effect of salt concentration on the cell. Figure 2A shows electrical vs. geometrical diameter change as a function of PBS concentration, where 100% refers to undiluted PBS. Figure 2B depicts the change in permittivity and geometric diameter using Earth Mover’s Distance (EMD) expressed as percentage change, as a function of the varying concentration of PBS.

[0029] Figure 3, comprising Figure 3A and 3B, depicts the results of example experiments demonstrating the response to Gefitinib by parental (DLDl.Par) and Gefitinib-resistant (DLDl.GefR) cell lines. Figure 3 A depicts the changes in the geometric diameter (x-axis) and electrical diameter (y-axis) using Earth Mover’s Distance (EMD), for the parental (DLD-l.Par, circle symbol) and gefitinib-resistant (DLDl.GefR, diamond symbol) cell-lines. The drug concentrations vary from 0 pM (No Drug) to 50 pM in a linear fashion as shown. For DLD 1.Par cell line, low concentrations of drug predominantly induce a volumetric change, whereas highAttorney Docket No. 206085-0196-00WOconcentrations cause structural change as picked up by the electrical diameter. In contrast, the effect of gefitinib on DLDl.GefR cells does not appear to be significant, as expected. Indeed, the induced changes are smaller than statistical uncertainty so long as the concentration is equal to or below 30 pM which is the level at which cells were maintained. The shades in figure markers indicate the cell viability values as detailed in colormap legend. Error bars indicate 2 standard deviation levels. Figure 3B depicts distance to the origin in the EMD plane for the two cell types as a function of drug concentrations (left y-axis), and the cell death percentage (i.e. 100 - cell viability). For each cell type, the total EMD distance runs parallel to the observed cell death levels

[0030] Figure 4, comprising Figures 4A and 4B, depicts the results of example experiments demonstrating the effect of cisplatin on gefitinib-resistant cell line (DLDl.GefR). Figure 4A depicts a comparison of the response of gefitinib-resistant cell line against two different drugs: gefitinib (blue) and cisplatin (red). When cisplatin is used, both the EMD response and the cell death values attain large values even at low concentrations; on the other hand, the response for gefitinib is smaller for both parameters, especially until 30 pM concentration of gefitinib. Figure 4B depicts a two-dimensional representation of changes in the cell lines as a function of drug. The gefitinib-resistant cell line (DLDl.GefR, triangles) responds to the drug by large shifts in geometric and electrical diameter. The effect of cisplatin on the parental DLD1 cell line (DLDl.Par, squares) is less pronounced, as discussed in the text. The drug concentration levels range from no drug at the origin to 50 pM at the end of the sequence, with 10 pM increments at each step. The shades in figure markers indicate the cell viability values as detailed in colormap legend. Error bars indicate 2 standard deviation levels. For ease of comparison, the figure also contains gefitinib treatments discussed in Figure 1A as grey symbols (circles: gefitinib treatment on DLDl.Par, diamonds: gefitinib treatment on DLDl.GefR).

[0031] Figure 5, comprising Figures 5A-5F depicts the results of tests on different cancer cell lines. Left Panels: Hotelling T2statistical test results (left-axis, bar plots) and viability values (right-axis, dotted-lines) for three different drugs and their corresponding parental (sensitive) and resistant cell lines, in orange and green respectively. Right Panels: Electric and geometric diameter distributions obtained by our sensor as a function of applied drug concentration. The tested drugs and cell lines are Gefitinib on DLD1 (Figures 5A and 5B), Cisplatin on HCC-1937Attorney Docket No. 206085-0196-00WO(Figures 5C and 5D), and Cisplatin on MDA-436 (Figures 5E and 5F). The larger Hotelling values indicate a large shift in the biophysical parameters in the cells. As evident from the figure, largest shifts in Hotelling occurs in the sensitive parental cell lines near the dose levels where there is a large drop in viability. The Hotelling T2values are calculated sequentially, i.e. each population is compared to the population at the previous (smaller) drug level. The Hotelling T2values in Figure 5C and 5D have initial large peaks close to their LCso value, and larger peaks later on at drug concentrations where the cell viability almost goes to zero.

[0032] Figure 6, comprising Figures 6A-6C, depicts measurements on cells isolated from PDOs. Figure 6A depicts the results of example experiments demonstrating the electrical vs geometrical diameter of cells. The gefitinib treated cells (orange) overlap mostly with the control group (green), while the bortezomib treated cells (purple) have smaller geometric diameters and slightly larger electrical diameters. Inset shows a general picture of organoids used in the experiments. Figure 6B depicts the results of example experiments demonstrating the Hotelling T2test indicates that the changes in the bortezomib treated group are much larger. Inset shows the geometric diameter distribution for the control (grey), gefitinib-treated (orange) and bortezomib-treated (purple) groups. Figure 6C depicts the geometric diameter distribution for the control (grey), gefitinib-treated (orange), and bortezomib-treated groups.

[0033] Figure 7, comprising Figures 7A-7H, depicts a schematic diagram representing an exemplary system and use (e.g., sensor architecture and operation). Figure 7A depicts a 3D render of the platform showing the microchannel spanning the inner and outer rings of the SRR, with the gold pads forming the Coulter counter also visible, and a simple diagram of the wiring scheme for each sensor. Figure 7B depicts a schematic view of an exemplary SRR / Coulter arrangement comprising a channel, a left electrode of the Coulter counter, a gold pad that is bonded to a right Coulter counter electrode, an outer SRR ring, and a sensing region containing a gap in an inner SRR ring. The inset depicts a close up of the gap and sensing region clearly showing an inner ring gap aligned with a constriction in the microfluidic channel and outer electrodes forming the Coulter counter and connected to the left electrode of the Coulter counter, and the gold pad bonded to the right Coulter counter electrode. Figures 7C-7H are a series of schematics showing a cell passing through the sensing region, and how its passage generates a signal response in both the current (Al) from the Coulter sensor and the capacitance change (AC)Attorney Docket No. 206085-0196-00WOfrom the microwave sensorthat represent geometrical, and electrical sizes respectively. As the cell nears the constriction, it blocks the current passing between the Coulter electrodes appearing as a reduction in conductivity. When the cell reaches the split-ring gap, it significantly alters the capacitance of the SRR structure especially that the electric field is concentrated at this point, therefore generating its own signal. Both those signals are then processed to calculate the electrical permittivity of the cell that passed through.

[0034] Figure 8 depicts the results of example experiments demonstrating the effect of cisplatin treatment, comparison of the parental DLD1 cell lines (blue, DLDl.Par) and gefitinib-resistant cell lines (green, DLDl.GefR) in terms of total EMD distance to the origin and cell death. Both parameters indicate that gefitinib-resistant cell line is more sensitive to cisplatin compared to the parental DLD1 cell line, as explained in the test.

[0035] Figure 9, comprising Figures 9A-9C, shows split-violin plots for the gefitinib treatment on the parental (DLDl.Par) and gefitinib-resistant (DLDl.GefR) DLD-1 cell lines, in geometric diameter (Figure 9A), electric diameter (Figure 9B), and dielectric permittivity (Figure 9C) dimensions.

[0036] Figure 10, comprising Figures 10A-10C, shows split-violin plots for the cisplatin treatment on the parental (DLDl.Par) and gefitinib-resistant (DLDl.GefR) DLD-1 cell lines, in geometric diameter (Figure 10A), electric diameter (Figure 10B), and dielectric permittivity (Figure 10C) dimensions.

[0037] Figure 11, comprising Figures 11A-11C, shows split-violin plots for the cisplatin treatment on the parental (HCC-1937.Par) and cisplatin-resistant (HCC-1937.CisR) HCC-1937 cell lines, in geometric diameter (Figure 11 A), electric diameter (Figure 1 IB), and dielectric permittivity (Figure 11C) dimensions.

[0038] Figure 12, comprising Figures 12A-12C, shows split-violin plots for the cisplatin treatment on the parental (MDA-436.Par) and cisplatin-resistant (MDA-436.CisR) MDA-436 cell lines, in geometric diameter (Figure 12A), electric diameter (Figure 12B), and dielectric permittivity (Figure 12C) dimensions.Attorney Docket No. 206085-0196-00WO

[0039] Figure 13 shows LC50 values indicating the development of drug resistance in the isogenic DLD-1 cell lines, as measured in a 96-well plate.

[0040] Figure 14 shows LC50 values indicating the development of drug resistance in the isogenic HCC-1937 cell lines, as measured in a 96-well plate.

[0041] Figure 15 shows LC50 values indicating the development of drug resistance in the isogenic MDA-436 (i.e. MDA-MB-436) cell lines, as measured in a 96-well plate.

[0042] Figure 16 is a diagram depicting a computer architecture.DETAILED DESCRIPTION OF THE INVENTION

[0043] It is to be understood that the figures and descriptions of the present invention have been simplified to illustrate elements that are relevant for a clearer comprehension of the present invention, while eliminating, for the purpose of clarity, many other elements found in systems and methods. Those of ordinary skill in the art may recognize that other elements and / or steps are desirable and / or required in implementing the present invention. However, because such elements and steps are well known in the art, and because they do not facilitate a better understanding of the present invention, a discussion of such elements and steps is not provided herein. The disclosure herein is directed to all such variations and modifications to such elements and methods known to those skilled in the art.

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.

[0045] As used herein, each of the following terms has the meaning associated with it in this section.Attorney Docket No. 206085-0196-00WO

[0046] The articles “a” and “an” are used herein to refer to one or to more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0047] The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. For example, as used herein, the singular forms “a”, “an” and “the” may be intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms “comprises,” “comprising,” “including,” and “having,” are inclusive and therefore specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. The method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed.

[0048] When an element or layer is referred to as being “on”, “engaged to”, “connected to” or “coupled to” another element or layer, it may be directly on, engaged, connected or coupled to the other element or layer, or intervening elements or layers may be present. In contrast, when an element is referred to as being “directly on,” “directly engaged to,” “directly connected to” or “directly coupled to” another element or layer, there may be no intervening elements or layers present. Other words used to describe the relationship between elements should be interpreted in a like fashion (e.g., “between” versus “directly between,” “adjacent” versus “directly adjacent,” etc ). As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0049] Although the terms first, second, third, etc., may be used herein to describe various elements, components, regions, layers and / or sections, these elements, components, regions, layers and / or sections should not be limited by these terms. These terms may be only used to distinguish one element, component, region, layer or section from another element, component, region, layer or section. That is, terms such as “first,” “second,” and other numerical terms, when used herein, do not imply a sequence or order unless clearly indicated by the context. Thus, a first element, component, region, layer or section discussed below could be termed a secondAttorney Docket No. 206085-0196-00WOelement, component, region, layer or section without departing from the teachings of the exemplary embodiments.

[0050] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, and ±0.1% from the specified value, as such variations are appropriate.

[0051] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0052] A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.

[0053] The phrase “therapeutically effective amount,” as used herein, refers to an amount that is sufficient or effective to prevent or treat (delay or prevent the onset of, prevent the progression of, inhibit, decrease or reverse) a disease or condition, including alleviating symptoms of such diseases.

[0054] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0055] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Where appropriate, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0056] The figures and descriptions provided herein may have been simplified to illustrate aspects that are relevant for a clear understanding of the herein described apparatuses, systems,Attorney Docket No. 206085-0196-00WOand methods, while eliminating, for the purpose of clarity, other aspects that may be found in typical similar devices, systems, and methods. Those of ordinary skill may thus recognize that other elements and / or operations may be desirable and / or necessary to implement the devices, systems, and methods described herein. But because such elements and operations are known in the art, and because they do not facilitate a better understanding of the present disclosure, for the sake of brevity a discussion of such elements and operations may not be provided herein.However, the present disclosure is deemed to nevertheless include all such elements, variations, and modifications to the described aspects that would be known to those of ordinary skill in the art.

[0057] Embodiments are provided throughout so that this disclosure is sufficiently thorough and fully conveys the scope of the disclosed embodiments to those who are skilled in the art. Numerous specific details are set forth, such as examples of specific components, devices, and methods, to provide a thorough understanding of embodiments of the present disclosure.Nevertheless, it will be apparent to those skilled in the art that certain specific disclosed details need not be employed, and that embodiments may be embodied in different forms. As such, the embodiments should not be construed to limit the scope of the disclosure. As referenced above, in some embodiments, well-known processes, well-known device structures, and well-known technologies may not be described in detail.Detailed Description

[0058] The present invention is based on the surprising discovery that the morphology of cancer cells changes in response to drugs to which the cancer cells are susceptible. For example, the geometric diameter and electrical diameter may increase or decrease when exposed to an anti-cancer drug depending on the mechanism of the drug. In contrast, cancer cells that are resistant to an anti-cancer drug remain unchanged.

[0059] In some embodiments, the invention relates to a system for measuring the geometric diameter and electrical diameter of a cell. In some embodiments, the system simultaneously measures the geometric diameter and electrical diameter of a cell. In some embodiments, the system measures the geometric diameter and electrical diameter of a single cell. In someAttorney Docket No. 206085-0196-00WOembodiments, the cell is a cancer cell. In some embodiments, the cell is a tumor cell. In some embodiments, the cell is obtained from a subject or patient.

[0060] In some embodiments, the invention relates to a method of measuring the geometric diameter and electrical diameter of a cell. In some embodiments, the method comprises simultaneously measuring the geometric diameter and electrical diameter of a cell. In some embodiments, the method comprises measuring the geometric diameter and electrical diameter of a single cell. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is a tumor cell. In some embodiments, the cell is obtained from a subject or patient.

[0061] In some embodiments, the invention relates to a method of identifying a cell or population of cells as susceptible or resistant to a drug. In some embodiments, the invention relates to a method of identifying a cell or population of cells as susceptible or resistant to a given dose of a drug. In some embodiments, the method comprises measuring the geometric diameter and electrical diameter of a cell. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is a tumor cell. In some embodiments, the cell is obtained from a subject or patient. In some embodiments, the drug is a cytostatic drug, a cytotoxic drug, or a mixed-effect drug.

[0062] In some embodiments, the invention relates to a method of treating cancer in a subject comprising identifying a population of cancer cells from the subject as being susceptible to a drug and administering that drug to the subject.Sy stem / D evice

[0063] As contemplated herein, the present invention includes a system for measuring the geometric diameter and electrical diameter of a cell.

[0064] Referring now to Figure 7, shown are schematic diagrams representing the sensor architecture and operation for a device or system 100 for measuring the geometric diameter and electrical diameter of a cell. Generally, system 100 comprises a body 102 (e.g., a wafer, a chip, a PDMS chip) with at least one microchannel 104 in or on the body 102, at least one least one split-ring resonator (SRR) 110 with concentric rings 112 positioned across each microchannel 104, and at least one Coulter counter 120 positioned within the SRR 110 and in proximity toAttorney Docket No. 206085-0196-00WOmicrochannel 102 for measuring samples (e.g., cells ) in the microchannel. In some examples, the SRR 110 is referred to as a “microwave sensor” or “high-frequency sensor, and the Coulter counter 120 is referred to as a “low-frequency sensor”.

[0065] Referring in particular now to Figure 7A, shown is a 3D render of the system 100 showing body 102 with microchannel 104 spanning an inner ring 112a and outer ring 112b of SRR 110, with a pair of electrodes 122 forming the at least one Coulter counter 120. The microchannel 104 comprises ports 106 disposed on opposing ends of the microchannel 104, such as an inlet 106a and an outlet 106b. It should be appreciated that fluid with samples (e.g., cells) may be flowed from inlet 106a to outlet 106b passing in proximity to SRR 110 and Coulter counter 120, for measuring the samples in the microchannel 104.

[0066] Also shown in Figure 7A is an exemplary wiring diagram for system 100 comprising a signal generator 150 and a first lock-in amplifier 160 electrically connected to outer ring 112b of SRR 110, and a second lock-in amplifier 170 electrically connected to the inner ring 112a of SRR 110 and the Coulter counter 120. In some embodiments, a vector network analyzer (VNA) is used to observe resonance characteristics of the SRR 110. System 100 may further comprise any number of pumps, reservoirs, tubings, and the like, fluidly connected with microchannel 104 via ports 106. Any portions of system 100 may be electronically and communicatively connected to a computing device (e.g., computer 1600 disclosed herein) for controlling and / or measuring aspects of the system. For example, signal generator 105 and lock-in amplifiers 160, 170 may be connected to a computing device for obtaining data or measurements from the system 100 and processing.

[0067] Referring in particular to Figure 7B, shown is a schematic view of an exemplary system 100 depicting an exemplary SRR 110 / Coulter counter 120 arrangement. In some embodiments, system 100 comprises at least one microchannel 102, a left electrode 122a and a right electrode 122b for Coulter counter 120. As shown, the left electrode 122a of Coulter counter 120 connects to first pad 124a, and right electrode 124b connects to pads 124b and 124c. In some embodiments, left electrode 122a electrically connects to a first pad 124a via a first lead 126a (e g., a trace), and right electrode 122b electrically connects to a second pad 124b via a second lead 126b, and a third pad 124c via a third lead 126c. The electrodes, leads and pads mayAttorney Docket No. 206085-0196-00WObe formed from any suitably conductive material, such as gold, silver, copper, or conductive polymers.

[0068] Aspects of the disclosure relate to the arrangement of microchannel 102, SRR 110 and Coulter counter 120. In some embodiments, microchannel 102 comprises a constriction 108 that narrows the microchannel 102 in a sensing region 130 of SRR 110 and Coulter counter 120. In some embodiments, constriction 108 is positioned to intersect or align with a portion of inner ring 112a of SRR 110. In some embodiments, inner ring 112a comprises a gap 114 in the ring, positioned to intersect with constriction 108 of microchannel 104. The gap 114 comprises a slotted portion 116 and a tabbed portion 118, the tabbed portion 118 partially extending into the slotted portion 116. In some embodiments, the tabbed portion 118 is asymmetrically positioned within the slotted portion 116.

[0069] The sensing region 130 comprises the gap 114 in inner ring 112b, wherein the inner ring 112b gap 114 is aligned with the constriction 108 in the microfluidic channel 104, and the electrodes 122 (e.g., left electrode 122a and right electrode 122b) form the Coulter counter 120 and ultimately connect to pads 124a and 124c, respectively. It should be appreciated that the disclosed sensor arrangement may be formed on any microfluidic device known by a person of ordinary level of skill in the art, and system 100 further may comprise any known microfluidic device features. Further, the disclosed system 100 and / or sensor arrangement may be used in conjunction with any known microfluidic devices or sensors.

[0070] Microchannel 104 may have any suitable length, width and height. For example, the length of microchannel 104 may range between 1 mm and 200 mm, and the width and height may range between 1 pm and 3 mm. In some embodiments, microchannel has a length of about 160 mm and a width of about 400 pm, and a height of about 250 pm. In some embodiments, constriction 108 has a width, length and / or height ranging between 1 pm and 1 mm. For example, in some embodiments, constriction 108 has a width of about 50 pm, a length of about 250 pm, and a height of about 250 pm.

[0071] The rings 112 of SRR 110 may be formed in any suitable size and shape. In some embodiments, the rings 112 of SRR 110 are round, or circular. Either of rings 112 may have a diameter, or major and minor diameters, ranging between 1 mm and 100 mm. For example, inAttorney Docket No. 206085-0196-00WOsome embodiments, inner ring 112a has a diameter of about 18 mm and outer ring 112b has a diameter of about 64 mm. In some embodiments, the diameter of outer ring 112b is greater than the diameter of inner ring 112a. The rings 112 may have at least one width ranging between 0.1 mm and 20 mm. For example, in some embodiments, inner ring 112a has a width of about 1 mm and outer ring has a width of about 4 mm. In some embodiments, the width of outer ring 112b is greater than the width of inner ring 112a. The rings 112 are concentric and a distance or offset exists between the rings. The distance or offset may range between 1 mm and 20 mm.

[0072] In some embodiments inner ring 112a comprises a tapering width that tapers from slotted portion 116 to tabbed portion 118. In some embodiments, slotted portion 116 has a width of about 250 pm. In some embodiments, tabbed portion 118 has a width of about 50 pm. In some embodiments, an overall width of slotted portion 116 is greater than an overall width of tabbed portion 118.

[0073] In some embodiments, one or more distances or gaps are formed between slotted portion 116 and tabbed portion 118. For example, a vertical distance and one or more lateral distances between the portions (measured along body 102). These distances or gaps may range between 1 pm and 1 mm. For example, in some embodiments, the vertical distance between slotted portion 116 and tabbed portion 118 is greater than the lateral distances. In some embodiments, one or more distances or gaps are about 50 pm. In some embodiments, the lateral distances or gaps between slotted portion 116 and tabbed portion 118 are asymmetrical, wherein a first lateral distance or gap is greater than a second lateral distance or gap. In some embodiments, a first lateral distance (e.g., to the left of tabbed portion 118) is 25 pm and a second lateral distance (e.g., to the right of tabbed portion 118) is 15 pm. In some embodiments, the first and second lateral distances range between 1 pm and 100 pm.

[0074] The rings 112 may have any thickness (measured upwards from body 102), or may be deposited on body 102 in any number of layers. For example, the thickness of rings 112 may range between 1 nm and 1 mm. In some embodiments, rings 112 have a thickness or layering of about 150 nm.

[0075] The electrodes 122 may be formed in any suitable size and shape. For examples, each electrode 122 may have a height (measured along body 102) or width ranging between 1Attorney Docket No. 206085-0196-00WOpm and 1 mm. The electrodes may have any thickness (measured upwards from body 102) or may be deposited on body 102 in any number of layers. For example, the thickness of electrodes 122 may range between 1 nm and 1 mm. The electrodes 122 may be separated by a distance along the body 102 or microchannel 104. For example, the electrodes 122 may be separated by a distance ranging between 1 nm and 1 mm. In some embodiments, electrodes 122 have a thickness or layering of about 150 nm. In some embodiments, body 102 comprises a fused silica wafer layered with a 100 nm thick gold layer.

[0076] Figures 7C-7H are a series of schematics showing a cell passing through the sensing region 130, and how its passage generates a signal response in both the current (Al) from the Coulter sensor 120 and the capacitance change (AC) from the microwave sensor (e.g., SRR 110) that represent geometrical, and electrical sizes respectively. As the cell nears the constriction 108, it blocks the current passing between the electrodes 122 appearing as a reduction in conductivity. When the cell reaches the split-ring gap 114, it significantly alters the capacitance of the SRR 110 structure especially that the electric field is concentrated at this point, therefore generating its own signal. Both those signals are then processed to calculate the electrical permittivity of the cell that passed through.

[0077] An exemplary system 100 fabrication is disclosed herein. In some embodiments, the system 100 comprises a planar SRR 110 design deposited on body 102 (e.g., fused silica wafers (Quartz Unlimited LLC)). Further details on system fabrication are described in detail in Tefek et al. (Tefek et al., 2023, Advanced Materials. 35:2304072; Secme et al., 2023, IEEE Sensors Journal. 23:6517-6529), the contents of which are incorporated by reference. In some embodiments, standard soft lithography is utilized to deposit a thin gold (Au) layer to form the SRR 110 using a pre-etched chromium (Cr) photolithography mask.

[0078] As discussed above, the SRR comprises of two concentric rings 112, one larger outer ring 112b, and another smaller inner ring 112a. The outer ring 112b is split, with two Au traces coming out of each end and terminating with a sub-miniature A (SMA) connector soldered onto the edge of the body 102 (e.g., fused-silica wafer). The inner ring 112a comprises another split circle where the split comprises a small region (e.g., sensing region 130) where signal sensing takes place. The sensing region 130 in the inner ring 112a comprises two asymmetrical gaps spanning a length of approximately 250 pm across (Figure 7B). Furthermore, two Au electrodesAttorney Docket No. 206085-0196-00WO122 are deposited surrounding the SRR 110 sensing region 130 and extend outwards for subsequent wire bonding. These electrodes 122 are connected to a low frequency signal generator 150 and act as a conventional Coulter counter 120. Body 102 comprises a straight (100 pm wide, 45 pm tall, and 70 mm long) microfluidic channel 104 with a constriction 108 (40 pm wide, 45 pm tall, 250 pm long) in the center developed in polydimethylsiloxane (PDMS, Dow Chemicals) comprising an inlet 106a and outlet 106b at either ends. Microchannel 104 is aligned and bonded (through O- plasma treatment) so that the constriction 108 in the channel covers the sensing region 130 of the SRR 110 precisely.

[0079] In some embodiments, the aforementioned system 100 may include one or more computing devices communicatively and / or operatively connected to the system for performing one or more steps of any of the disclosed methods. In some embodiments, the disclosed system and methods thereof may utilize machine learning (ML) and artificial intelligence (Al) to process any obtained data or measurements. Accordingly, the disclosed system and methods thereof may operate at least in part with one or more AI / ML algorithms, engines and / or modules that may at least partially reside on a computing device (e.g., computer 1600 disclosed herein) and function with one or more networks (e.g., neural networks).

[0080] In some aspects of the present invention, software executing the instructions provided herein may be stored on a non-transitory computer-readable medium, wherein the software performs some or all of the steps of the present invention when executed on a processor.

[0081] Aspects of the invention relate to algorithms executed in computer software. Though certain embodiments may be described as written in particular programming languages, or executed on particular operating systems or computing platforms, it is understood that the system and method of the present invention is not limited to any particular computing language, platform, or combination thereof. Software executing the algorithms described herein may be written in any programming language known in the art, compiled or interpreted, including but not limited to C, C++, C#, Objective-C, Java, JavaScript, MATLAB, Python, PHP, Perl, Ruby, or Visual Basic. It is further understood that elements of the present invention may be executed on any acceptable computing platform, including but not limited to a server, a cloud instance, a workstation, a thin client, a mobile device, an embedded microcontroller, a television, or any other suitable computing device known in the art.Attorney Docket No. 206085-0196-00WO

[0082] Parts of this invention are described as software running on a computing device. Though software described herein may be disclosed as operating on one particular computing device (e.g. a dedicated server or a workstation), it is understood in the art that software is intrinsically portable and that most software running on a dedicated server may also be run, for the purposes of the present invention, on any of a wide range of devices including desktop or mobile devices, laptops, tablets, smartphones, watches, wearable electronics or other wireless digital / cellular phones, televisions, cloud instances, embedded microcontrollers, thin client devices, or any other suitable computing device known in the art.

[0083] Similarly, parts of this invention are described as communicating over a variety of wireless or wired computer networks. For the purposes of this invention, the words “network”, “networked”, and “networking” are understood to encompass wired Ethernet, fiber optic connections, wireless connections including any of the various 802.11 standards, cellular WAN infrastructures such as 3G, 4G / LTE, or 5G networks, Bluetooth®, Bluetooth® Low Energy (BLE) or Zigbee® communication links, or any other method by which one electronic device is capable of communicating with another. In some embodiments, elements of the networked portion of the invention may be implemented over a Virtual Private Network (VPN).

[0084] Figure 16 and the following discussion are intended to provide a brief, general description of a suitable computing environment in which the invention may be implemented. While the invention is described above in the general context of program modules that execute in conjunction with an application program that runs on an operating system on a computer, those skilled in the art will recognize that the invention may also be implemented in combination with other program modules.

[0085] Generally, program modules include routines, programs, components, data structures, and other types of structures that perform particular tasks or implement particular abstract data types. Moreover, those skilled in the art will appreciate that the invention may be practiced with other computer system configurations, including hand-held devices, multiprocessor systems, microprocessor-based or programmable consumer electronics, minicomputers, mainframe computers, and the like. The invention may also be practiced in distributed computing environments where tasks are performed by remote processing devices that are linked through aAttorney Docket No. 206085-0196-00WOcommunications network. In a distributed computing environment, program modules may be located in both local and remote memory storage devices.

[0086] Figure 16 depicts an illustrative computer architecture for a computer 1600 for practicing the various embodiments of the invention. The computer architecture shown in Figure 16 illustrates a conventional personal computer, including a central processing unit 1650 (“CPU”), a system memory 1605, including a random access memory 1610 (“RAM”) and a readonly memory (“ROM”) 1615, and a system bus 1635 that couples the system memory 1605 to the CPU 1650. A basic input / output system containing the basic routines that help to transfer information between elements within the computer, such as during startup, is stored in the ROM 1615. The computer 1600 further includes a storage device 1620 for storing an operating system 1625, application / program 1630, and data.

[0087] The storage device 1620 is connected to the CPU 1650 through a storage controller (not shown) connected to the bus 1635. The storage device 1620 and its associated computer-readable media provide non-volatile storage for the computer 1600. Although the description of computer-readable media contained herein refers to a storage device, such as a hard disk or CD-ROM drive, it should be appreciated by those skilled in the art that computer-readable media can be any available media that can be accessed by the computer 1600.

[0088] By way of example, and not to be limiting, computer-readable media may comprise computer storage media. Computer storage media includes volatile and non-volatile, removable and non-removable media implemented in any method or technology for storage of information such as computer-readable instructions, data structures, program modules or other data.Computer storage media includes, but is not limited to, RAM, ROM, EPROM, EEPROM, flash memory or other solid state memory technology, CD-ROM, DVD, or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store the desired information and which can be accessed by the computer.

[0089] According to various embodiments of the invention, the computer 1600 may operate in a networked environment using logical connections to remote computers through a network 1640, such as TCP / IP network such as the Internet or an intranet. The computer 1600 mayAttorney Docket No. 206085-0196-00WOconnect to the network 1640 through a network interface unit 1645 connected to the bus 163 . It should be appreciated that the network interface unit 1645 may also be utilized to connect to other types of networks and remote computer systems.

[0090] The computer 1600 may also include an input / output controller 1655 for receiving and processing input from a number of input / output devices 1660, including a keyboard, a mouse, a touchscreen, a camera, a microphone, a controller, a joystick, or other type of input device. Similarly, the input / output controller 1655 may provide output to a display screen, a printer, a speaker, or other type of output device. The computer 1600 can connect to the input / output device 1660 via a wired connection including, but not limited to, fiber optic, Ethernet, or copper wire or wireless means including, but not limited to, Wi-Fi, Bluetooth, NearField Communication (NFC), infrared, or other suitable wired or wireless connections.

[0091] As mentioned briefly above, a number of program modules and data files may be stored in the storage device 1620 and / or RAM 1610 of the computer 1600, including an operating system 1625 suitable for controlling the operation of a networked computer. The storage device 1620 and RAM 1610 may also store one or more applications / programs 1630. In particular, the storage device 1620 and RAM 1610 may store an application / program 1630 for providing a variety of functionalities to a user. For instance, the application / program 1630 may comprise many types of programs such as a word processing application, a spreadsheet application, a desktop publishing application, a database application, a gaming application, internet browsing application, electronic mail application, messaging application, and the like. According to an embodiment of the present invention, the application / program 1630 comprises a multiple functionality software application for providing word processing functionality, slide presentation functionality, spreadsheet functionality, database functionality and the like.

[0092] The computer 1600 in some embodiments can include a variety of sensors 1665 for monitoring the environment surrounding and the environment internal to the computer 1600. These sensors 1665 can include a Global Positioning System (GPS) sensor, a photosensitive sensor, a gyroscope, a magnetometer, thermometer, a proximity sensor, an accelerometer, a microphone, biometric sensor, barometer, humidity sensor, radiation sensor, or any other suitable sensor.Attorney Docket No. 206085-0196-00WO

[0093] Aspects of the disclosure relate to machine learning executed on a computing device (e.g., computer 1600). In some embodiments, the disclosed system and method utilize ML / Al algorithms and / or models, including one or more neural networks, that may operate on at least one computing device (e.g., computer 1000). The disclosed system may employ various types of neural networks known in the art, including but not limited to feedforward neural networks (FNNs), convolutional neural networks (CNNs), recurrent neural networks (RNNs), transformer networks, autoencoders, generative adversarial networks (GANs), Radial Basis Function Networks (RBFNs), extreme learning machines (ELMs), quantum neural networks (QNNs), and deep neural networks (DNNs).

[0094] ML is a branch of Al that enables systems to learn and improve from experience without being explicitly programmed. ML models analyze data sets to identify patterns and correlations, and then use those patterns to make predictions or decisions. ML models can generally be categorized into three primary types: supervised learning, unsupervised learning, and semi-supervised learning.

[0095] Supervised learning involves training a model using labeled datasets to classify data or predict outcomes accurately. As input data is fed into the model, the model adjusts its internal parameters (e.g., weights) to minimize prediction errors. Common methods used in supervised learning include neural networks, naive Bayes classifiers, linear regression, logistic regression, random forests, and support vector machines (SVMs).

[0096] Classification is a common task in supervised learning, where data inputs are categorized into distinct classes. Classification models may include binary classifiers and multiclass classifiers. A decision tree is a widely used classification method that applies a sequence of "if-then" conditions to narrow down possible outcomes.

[0097] Regression is another form of supervised learning where the output is a continuous variable rather than a discrete category. Linear regression predicts a continuous value based on a linear relationship between inputs and outputs, while logistic regression predicts categorical outcomes based on defined inputs.

[0098] Unsupervised learning involves analyzing unlabeled datasets to identify hidden patterns or groupings without human intervention. Principal component analysis (PCA) andAttorney Docket No. 206085-0196-00WOsingular value decomposition (SVD) are common techniques used to reduce data dimensionality and reveal underlying structures.

[0099] Clustering is a key unsupervised learning technique where data points are grouped based on shared features or proximity. K-means clustering is a widely used method where the number of clusters is defined by a variable "k," and the algorithm iteratively adjusts cluster centroids to minimize variance within each cluster. Other clustering methods include hierarchical clustering and probabilistic clustering.

[0100] Semi-supervised learning combines elements of both supervised and unsupervised learning. A model is initially trained using a smaller labeled dataset, which then guides the classification and feature extraction from a larger unlabeled dataset. Semi-supervised learning is particularly useful when acquiring large amounts of labeled data is costly or impractical.

[0101] Deep learning is a subfield of ML that uses neural networks with multiple hidden layers to process and analyze complex data. Neural networks mimic the structure and function of the human brain, comprising layers of interconnected nodes (neurons). Each neuron receives input data, applies a transformation based on assigned weights, and passes the result to the next layer.

[0102] A typical neural network consists of: input layer - receives raw data inputs; hidden layer(s) - applies mathematical transformations using weighted connections; and output layer -generates the final prediction or classification.

[0103] Convolutional neural networks (CNNs) are a type of neural network particularly well-suited for processing image and spatial data. CNNs use convolutional layers to extract spatial features from input data, pooling layers to reduce dimensionality, and fully connected layers to generate output predictions.

[0104] Deep neural networks (DNNs) are composed of multiple hidden layers and are capable of learning complex patterns in large datasets. Recurrent neural networks (RNNs) are a type of deep learning network designed for sequential data, such as time series or natural language, where previous inputs influence future outputs. Long short-term memory (LSTM) networks are a specialized form of RNN that mitigates issues with long-term dependencies.Attorney Docket No. 206085-0196-00WO

[0105] Generative Adversarial Networks (GANs) are a class of ML models in which two neural networks — a generator and a discriminator — are trained together in a competitive framework. The generator creates synthetic data (e.g., images, audio) from random noise, while the discriminator evaluates whether the generated data is real or fake. The generator improves its output by trying to fool the discriminator, while the discriminator becomes better at distinguishing real data from generated data. This adversarial process drives both networks to improve over time, leading to the generation of highly realistic data.

[0106] Types of GANs include: Vanilla GAN - The original GAN model, where the generator and discriminator are trained using a minimax loss function. Deep Convolutional GAN (DCGAN) - Uses convolutional layers instead of fully connected layers, improving the quality of generated images. Conditional GAN (cGAN) - Conditions the generation process on class labels or other input data, enabling targeted generation (e.g., generating images of specific objects). Wasserstein GAN (WGAN) - Introduces the Wasserstein distance (Earth Mover’s distance) as the loss function, which improves training stability and reduces mode collapse (when the generator produces limited variations of data). WGAN-GP (Wasserstein GAN with Gradient Penalty) - Improves WGAN by adding a gradient penalty to enforce the Lipschitz constraint, further enhancing training stability. CycleGAN - Used for unpaired image-to-image translation (e.g., converting paintings to photos) by enforcing consistency between the forward and backward transformations. StyleGAN - Generates high-resolution and highly detailed images using a style-based generator architecture, allowing greater control over features like face shape and texture. GANs are widely used in fields such as computer vision, natural language processing, and creative design, but they can be difficult to train due to instability and mode collapse — challenges that models like WGAN and WGAN-GP address effectively.

[0107] In some embodiments, the disclosed system may include an Al model trained using reinforcement learning, where an agent learns to make decisions through trial and error by interacting with an environment and receiving feedback in the form of rewards or penalties. It should be appreciated that the disclosed systems and methods may utilize any of the components and features of computer 1600, including any of the features, techniques and processing steps disclosed for Al and ML.Attorney Docket No. 206085-0196-00WOMethods of Measurement

[0108] In some embodiments, the invention relates to a method of measuring the geometric diameter and electrical diameter of a cell. In some embodiments, the method comprises measuring the geometric diameter and electrical diameter of a single cell. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is a tumor cell.

[0109] An exemplary method of using system 100 is disclosed herein. Liquid containing samples are delivered through the microfluidic channel 104 using a Fluigent (MFCS-EZ) pressure control system. Typical flow rates ranged from 8 to 15 pL / min with a cell transit time through the SRR 110 sensing region 130 being between 10-20 ms.

[0110] The system 100 comprises a low-frequency (e.g., Coulter counter 120) and a high-frequency (microwave) sensor (e.g., SSR 110) to obtain the internal property of the cells. In the low-frequency part, referred as Coulter counter 120, an insulating particle’s passage results in partial blockage of ionic current being conducted between two electrodes 122, the decrease in current being proportional to the geometrical volume of the particle. A 0.5 Vpp signal at 0.5 MHz from lock-in amplifier 170 (Zurich Instruments, HF2LI) is used to drive one electrode 122b while the resulting current flowing through the ionic solution is collected from the other electrode 122a. This signal is converted to voltage by a transimpedance amplifier (Zurich Instruments, HF2TA) and consequently read out by the lock-in amplifier 160.

[0111] The high-frequency part of the sensor comprises an SRR 110 for electrical size measurement of the cells. The SRR 110 comprises two concentric rings 112, the inner ring 112a is excited inductively by the microwave signal fed through the outer ring 112b. A highly concentrated electric field is created in the gap 114 (split region) of the inner ring 112a due to the standing wave mode (Abduljabar et al., 2014, IEEE Transactions on Microwave Theory and Techniques. 62:679-688; Lee & Yook, 2008, Applied Physics Letters. 92; Salim & Lim et al., 2016, Sensors. 16:1802) and passage of particle through the gap 114 cause phase and amplitude change of the resonator due to capacitance change in the sensing region 130 representative of the particle’s geometric volume and the permittivity. In this specific design, time delay of the signals from two lateral gaps of unequal width (15 and 25 pm) between slotted portion 116 and tabbedAttorney Docket No. 206085-0196-00WOportion 118 in the inner ring 112a are utilized to obtain the height information of the passing particles and for calibrating the signal (Figure 7).

[0112] In some embodiments, a vector network analyzer (VNA) is used to observe resonance characteristics of the SRR 110 before connecting to the custom measurement circuitry for conducting measurements (Tefek et al., 2023, Advanced Materials. 35:2304072). A signal generator 150 is used as a signal source and its frequency is set as the resonance frequency of the SRR 110 (=5.3 GHz). A 0.5 Vpp signal at the resonance frequency is fed to the SRR 110. Two lock-in amplifiers 160, 170 (Zurich Instruments, MFLI) are used to employ single side band modulation (SSBM) as part of the measurement circuitry. A custom-built single side band (SSB) heterodyne circuitry (Ferrier et al, 2009, Lab on a Chip. 9:3406-3412; Nikolic-Jaric et al., 2009, Biomicrofluidics. 3; Kelleci et al., 2018, Lab on a Chip. 18:463-472) is used as the resonance frequency is higher than the maximum operational frequency of the lock-in amplifiers 160, 170. Up-conversion and down-conversion is utilized for digitally reading the phase and amplitude of the signal. To obtain signals from fast moving cells, the time constant of the lock-in amplifiers 160, 170 is set as 501 ps, and the sampling rates are set as 14.39 kSa / s and 13.39 KSa / s for low-frequency sensing, and high-frequency sensing, respectively.

[0113] Signal obtained from the low-frequency part of the sensor provides the cell’s geometrical volume upon height calibration by the use of calibration particles (Polystyrene 20 pm, Sigma-Aldrich) added into the sample for this purpose. The phase and amplitude response of the microwave resonator are obtained from the high-frequency part of the sensor, and the out-of-phase component (Y = R sinO) of the reflected voltage, is calculated which probes any minute change in the capacitance of the resonator, as described in Equation 1 of the examples section below.

[0114] In Equation 1, AY refers to the change in the out-of-phase component of the reflected voltage, C refers to the total capacitance of the resonator, and AC refers to the change of capacitance due to the passage of a cell. The change of phase (A9) and amplitude (AR) are extracted fortransit of single cells through the sensing region 130. The capacitance change due to the presence of a cell can be calculated by Equation 2 of the examples section below, where R and 0 refer to the baseline amplitude and phase values in raw signal.Attorney Docket No. 206085-0196-00WO

[0115] The capacitance change calculated is a function of geometrical volume and the Clausius-Mosotti factor of the cell (a factor depending on the microwave permittivity values of the cell and the medium), decoupling the geometrical volume as obtained by the Coulter counter 120 enables attaining the microwave permittivity of individual cells. The details of the circuitry, calibration procedure, and data analysis can be found in Tekek et al. (Tefek et al., 2023, Advanced Materials. 35:2304072), the contents of which are incorporated by reference. The Earth-Mover Distance calculations follow the standard definition (Kimmerling et al., 2022, Communications Biology. 5:1295; Orlova et al., 2016, PLoS One. 11 :e0151859) and the error bars are calculated using bootstrapping techniques using 200 resamples.

[0116] In some embodiments, the method comprises isolating a cell or population of cells from a subject. In some embodiments, the method comprises isolating a cell or population of cells from a tumor from a subject. In some embodiments, the method comprises culturing a population of cells from a sample of cells from a subject. In some embodiments, the method comprises obtaining cells from a patient-derived organoid (PDO).

[0117] Cells for analysis using the device and methods of the invention may be obtained from a subject or patient. Methods of obtaining cell-containing samples from a subject or patient are well-known to those of skill in the art and include, but are not limited to, aspirations or drawing of blood or other fluids. Samples may include, but are not limited to, blood, lymph, urine, gynecological fluids, biopsies, amniotic fluid and smears. In one embodiment, a sample is a bone marrow aspirate. In one embodiment, the sample is a biopsy sample. Samples may be obtained from a patient by a variety of techniques including, for example, by scraping or swabbing an area or by using a needle to aspirate bodily fluids. In some embodiments the sample may be treated or processed by methods known in the art (e.g., centrifugation).

[0118] In some embodiments, the cell is isolated from a subject having or at risk for cancer. The following are non-limiting examples of cancers from which tumor cells may be isolated for the disclosed methods: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, appendix cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and spinal cord tumors, brain stem glioma, brain tumor, breast cancer, bronchial tumors, burkitt lymphoma, carcinoid tumor, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumors, central nervous system lymphoma, cerebellarAttorney Docket No. 206085-0196-00WOastrocytoma, cerebral astrocytoma / m align ant glioma, cerebral astrocytotna / malignant glioma, cervical cancer, childhood visual pathway tumor, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous cancer, cutaneous t-cell lymphoma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, ewing family of tumors, extracranial cancer, extragonadal germ cell tumor, extrahepatic bile duct cancer, extrahepatic cancer, eye cancer, fungoides, gallbladder cancer, gastric (stomach) cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (gist), germ cell tumor, gestational cancer, gestational trophoblastic tumor, glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, histiocytosis, hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, hypothalamic tumor, intraocular (eye) cancer, intraocular melanoma, islet cell tumors, kaposi sarcoma, kidney (renal cell) cancer, langerhans cell cancer, langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocvtoma of bone and osteosarcoma, medulloblastoma, medulloepithelioma, melanoma, merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, myelogenous leukemia, myeloid leukemia, myeloma, myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal parenchymal tumors of intermediate differentiation, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, primary central nervous system cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter cancer, respiratory tract carcinoma involving the nut gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, sezary syndrome, skin cancer (melanoma),Attorney Docket No. 206085-0196-00WOskin cancer (nonmelanoma), skin carcinoma, small cell lung cancer, small intestine cancer, soft tissue cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, stomach (gastric) cancer, supratentorial primitive neuroectodermal tumors, supratentorial primitive neuroectodermal tumors and pineoblastoma, T-cell lymphoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer, transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, waldenstrom macroglobulinemia, and wilms tumor.

[0119] In some embodiments, the method comprises measuring electrical diameter and geometric diameter of the cell at the microwave band using the device described herein. In some embodiments, dielectric permittivity (e) at the microwave band (microwave permittivity) is derived by normalizing the electrical diameter to the geometric diameter.

[0120] In some embodiments, the method comprises monitoring biophysical changes in a cell. In some embodiments, the method comprises assaying electrical diameter and geometric diameter of a cell exposed to a drug.

[0121] In some embodiments, the invention provides a method for identifying the mechanism of action of a drug by measuring the change in electrical diameter and geometric diameter of a cell exposed to the drug.Methods of Identifying Drug Susceptibility

[0122] In some embodiments, the invention relates to a method of identifying a cell as susceptible or resistant to a drug or a given dose of a drug. In some embodiments, the method comprises measuring the geometric diameter and electrical diameter of a cell. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is a tumor cell. In some embodiments, the drug is an anti-cancer drug. In some embodiments, the drug is a cytostatic drug, a cytotoxic drug, or a mixed-effect drug.

[0123] In certain embodiments, the method provides for personalized evaluation regarding whether a subject or patient would be responsive to a specific drug treatment. For example, in certain embodiments, the method comprises obtaining one or more cells from the subject orAttorney Docket No. 206085-0196-00WOpatient, for example one or more cancer or tumor cells of the subject or patient, and evaluating the effect of the drug using the method and system described herein.

[0124] In certain embodiments, the method provides for an evaluation of a drug candidate to evaluate the effect of the drug candidate on a cancer or tumor cell. For example, a drug candidate of interest or a candidate from a library can be evaluated using the method and system described herein. The cells to evaluate a drug candidate can comprise cells from a specific patient or subject, or model tumor cells (e.g. cell lines).

[0125] In some embodiments, the method comprises obtaining a cell or population of cells from a subject as described elsewhere herein. In some embodiments, the method comprises establishing a “baseline” measurement of the cell or population of cells. In some embodiments, the baseline measurement comprises the geometric diameter and electrical diameter of the cell or population of cells under normal conditions (i.e., without being exposed to a drug). Therefore, in some embodiments, the method comprises measuring the geometric diameter and electrical diameter of the cell or population of cells under normal conditions as described elsewhere herein.

[0126] In some embodiments, the method comprises exposing the cell or population of cells to a drug and subsequently measuring the geometric diameter and electrical diameter. In some embodiments, the method comprises exposing the cell or population of cells to a specific dose of a drug and subsequently measuring the geometric diameter and electrical diameter. In some embodiments, the method comprises comparing the average geometric diameter and electrical diameter of the population of cells with and without exposure to a drug.

[0127] In some embodiments, a cell or population of cells is determined to be susceptible to a drug or a given dose of a drug if the geometric diameter is changed when exposed to the drug compared to the baseline measurement. In some embodiments, a cell or population of cells is determined to be susceptible to a drug if the geometric diameter increases by at least 5%, by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, or by at least 60%, by at least 70%, by at least 80%, by at least 90%, or by at least 100% compared to the baseline measurement. In some embodiments, a cell or population of cells is determined to be susceptible to a drug if the geometric diameter decreases by at least 5%, by at least 10%, by at least 20%, byAttorney Docket No. 206085-0196-00WOat least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, or by at least 100% compared to the baseline measurement.

[0128] In some embodiments, a cell or population of cells is determined to be susceptible to a drug or a given dose of a drug if the electrical diameter is changed when exposed to the drug compared to the baseline measurement. In some embodiments, a cell or population of cells is determined to be susceptible to a drug if the electrical diameter increases by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, or by at least 100% compared to the baseline measurement. In some embodiments, a cell or population of cells is determined to be susceptible to a drug if the electrical diameter decreases by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, or by at least 100% compared to the baseline measurement.

[0129] In some embodiments, a cell or population of cells is determined to be susceptible to a drug if the geometric diameter and the electrical diameter are both changed when compared with the baseline measurement.

[0130] In some embodiments, a cell or population of cells is determined to be resistant to a drug if the geometric diameter remains unchanged when compared with the baseline measurement. In some embodiments, a cell or population of cells is determined to be resistant to a drug if the electrical diameter remains unchanged when compared with the baseline measurement.Methods of Treatment

[0131] In some embodiments, the invention provides a method of treating cancer in a subject. In some embodiments, the method comprises treating or preventing metastasis of cancer in a subject.

[0132] In some embodiments, the method comprises isolating cancer cells from a subject. In some embodiments, the method comprises identifying a cell as susceptible or resistant to a drug. In some embodiments, the method comprises measuring the geometric diameter and electrical diameter of a cell.Attorney Docket No. 206085-0196-00WO

[0133] In some embodiments, the method comprises identifying cancer cells from a subject as being susceptible to a drug and administering that drug to the subject. For example, in some embodiments, the method comprises identifying cancer cells from a subject as being susceptible to a cytostatic drug and administering a cytostatic drug to the subject. In some embodiments, the method comprises identifying cancer cells from a subject as being susceptible to a cytotoxic drug and administering a cytotoxic drug to the subject. In some embodiments, the method comprises identifying cancer cells from a subject as being susceptible to a mixed-effect drug and administering a mixed-effect drug to the subject.

[0134] In some embodiments, the method comprises identifying cancer cells from a subject as being susceptible to gefitinib and administering gefitinib to the subject. In some embodiments, the method comprises identifying cancer cells from a subject as being susceptible to cisplatin and administering cisplatin to the subject. In some embodiments, the method comprises identifying cancer cells from a subject as being susceptible to bortezomib and administering bortezomib to the subject. In some embodiments, the method comprises identifying cancer cells from a subject as being susceptible to tamoxifen and administering tamoxifen to the subject.

[0135] Anti-cancer drugs that can be used include, but are not limited to, acivicin, aclarubicin, acodazole hydrochloride, acronine, adozelesin, aldesleukin, altretamine, ambomycin, ametantrone acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate, bizelesin, bleomycin sulfate, brequinar sodium, bropirimine, busulfan, cactinomycin, calusterone, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefingol, chlorambucil, cirolemycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine, dexormaplatin, dezaguanine, dezaguanine mesylate, diaziquone, docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, duazomycin, edatrexate, eflornithine hydrochloride, elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin hydrochloride, erbulozole, erlotinib, esorubicin hydrochloride, estramustine, estramustine phosphate sodium, etanidazole, etoposide, etoposide phosphate, etoprine, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, fluorocitabine, fosquidone, fostriecin sodium, fulvestrant, gefitinib,Attorney Docket No. 206085-0196-00WOgemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, ilmofosine, interleukin II (including recombinant interleukin II, or rIL2), interferon alfa-2a, interferon alfa-2b, interferon alfa-nl, interferon alfa-n3, interferon beta-I a, interferon gamma-I b, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, liarozole hydrochloride, lometrexol sodium, lomustine, losoxantrone hydrochloride, masoprocol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium, metoprine, meturedepa, mitindomide, mitocarcin, mitocromin, mitogillin, mitomalcin, mitomycin, mitosper, mitotane, mitoxantrone hydrochloride, mycophenolic acid, nocodazole, nogalamycin, ormaplatin, oxisuran, paclitaxel, pegaspargase, peliomycin, pentamustine, peplomycin sulfate, perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, plomestane, porfimer sodium, porfiromycin, prednimustine, procarbazine hydrochloride, puromycin, puromycin hydrochloride, pyrazofurin, riboprine, rogletimide, sacituzumab govitecan (SG), safingol, safingol hydrochloride, semustine, simtrazene, sparfosate sodium, sparsomycin, spirogermanium hydrochloride, spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenur, T-DM1 talisomycin, tamoxifen, tecogalan sodium, tegafur, teloxantrone hydrochloride, temoporfm, teniposide, teroxirone, testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, toremifene citrate, trastuzumab, trastuzumab deruxtecan (T-DXD), trestolone acetate, triciribine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin, tubulozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfm, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate sulfate, vinleurosine sulfate, vinorelbine tartrate, vinrosidine sulfate, vinzolidine sulfate, vorozole, zeniplatin, zinostatin, zorubicin hydrochloride.AI / ML Methods

[0136] Aspects of the disclosure relate to augmenting system 100 and the methods thereof with AI / ML for processing, classifying and making predictions and recommendations based on the obtained data or measurements. In some embodiments, the disclosed systems and methods further employ Al and / or ML models configured to analyze raw and derived measurements or data obtained from system 100 and any associated methods. Non-limiting examples of data that may be captured and provided as inputs to an ML model include: time-series Coulter counterAttorney Docket No. 206085-0196-00WOsignals (e.g., Al traces), microwave sensor responses (e.g., phase and amplitude changes, AC, AY), geometric diameter, electrical diameter, and derived microwave permittivity values for individual cells; population-level statistics such as distributions of geometric / electrical diameters, permittivity histograms, Hotelling T2 values, Earth Mover’s Distance (EMD) values, and LC50 measurements; and contextual metadata including drug identity, drug class (e.g., cytostatic, cytotoxic, mixed-effect), concentration, exposure time, cell line identity (e.g., parental vs resistant), PDO identifier, cancer type, and prior treatment history for the subject. In some embodiments, training data for an AI / ML model may comprise large cohorts of such measurements paired with ground-truth clinical or experimental labels, including known drug susceptibility or resistance calls, measured cell viability, observed mechanisms of cell death, time to progression, or patient-level treatment outcomes, thereby enabling supervised or semisupervised learning on heterogeneous biophysical and clinical feature sets.

[0137] In some embodiments, one or more trained AI / ML models are configured to automatically classify a cell or population of cells as susceptible or resistant to one or more drugs based on the multidimensional pattern of geometric diameter, electrical diameter, permittivity, and their evolution across dose and time, optionally in combination with viability and LC50 data. In some embodiments, the models may further predict the likelihood, timing, or trajectory of emerging drug resistance under a given regimen, recommend an alternative drug or drug combination expected to yield a desired biophysical response profile (e.g., specific shifts in geometric and / or electrical diameter consistent with effective cytotoxicity), or rank candidate drugs and dosing schedules for a given subject or PDO in order of predicted efficacy. In some embodiments, AI / ML models may be configured to infer or suggest a mechanism of action or cell death pathway (e.g., apoptotic versus non-apoptotic signatures) based on characteristic changes in the measured parameters, and to output one or more treatment-guiding recommendations, such as indicating a preferred drug class, a preferred concentration range, a sequence of drugs to test, and / or an alert that a currently effective drug is likely to lose efficacy due to early biophysical signatures of resistance.EMBODIMENTSAttorney Docket No. 206085-0196-00WO

[0138] The invention may comprise or consist of any one of the following embodiments, or any combination thereof.

[0139] 1. A system for measuring the geometric diameter and electrical diameter of a cell, comprising:a planar body with a microchannel comprising an inlet and an outlet, wherein the microchannel comprises a centrally located constriction;a split-ring resonator (SRR) positioned on the body comprising an inner ring and an outer ring, wherein the inner ring comprises a gap in the ring; anda Coulter counter positioned on the body comprising first and second electrodes,wherein the constriction of the microchannel, the gap of the inner ring, and the first and second electrodes of the Coulter counter are aligned to form a sensing region for measuring a sample comprising at least one cell.

[0140] 2. The system of embodiment 1, wherein the inner ring comprises a first end comprising a tabbed portion, and a second end comprising a slotted portion, wherein the tabbed portion at least partially extends into the slotted portion to form the gap.

[0141] 3. The system of embodiment 2, wherein the tabbed portion is asymmetrically positioned in the slotted portion.

[0142] 4. The system of embodiment 3, wherein the width of the inner ring tapers from the second end to the first end.

[0143] 5. The system of embodiment 1, wherein the width of the inner ring is less than the width of the outer ring.

[0144] 6. The system of embodiment 1, wherein the first and second electrodes of the Coulter counter are separated by a distance along the length of the body ranging between 10 pm and 1000 pm.Attorney Docket No. 206085-0196-00WO

[0145] 7. The system of embodiment 1, further comprising a signal generator and a first lock-in amplifier electrically connected to the outer ring of the SRR, and a second lock-in amplifier electrically connected to the electrodes of the Coulter counter.

[0146] 8. The system of embodiment 1, wherein the SRR is a high-frequency sensor and the Coulter counter is a low-frequency sensor.

[0147] 9. The system of embodiment 1, wherein the sample comprising at least one cell is passed from the inlet to the outlet across the sensing region to measure the geometric diameter and electrical diameter of the at least one cell.

[0148] 10. A method of measuring the geometric diameter and electrical diameter of a sample comprising at least one cell using the system of embodiment 1.

[0149] 11. A method of identifying a population of cells as susceptible or resistant to a drug comprising measuring the geometric diameter and electrical diameter of the at least one cell.

[0150] 12. The method of embodiment 11, wherein the method comprises exposing the at least one cell to a given dose of the drug and subsequently measuring the geometric diameter and electrical diameter of the at least one cell.

[0151] 13. The method of embodiment 12, wherein the method comprises:a) providing a system comprising:a planar body with a microchannel comprising an inlet and an outlet, wherein the microchannel comprises a centrally located constriction;a split-ring resonator (SRR) positioned on the body comprising an inner ring and an outer ring, wherein the inner ring comprises a gap in the ring; and a Coulter counter positioned on the body comprising first and second electrodes, wherein the constriction of the microchannel, the gap of the inner ring, and the first and second electrodes of the Coulter counter are aligned to form a sensing region; andb) contacting the at least one cell to the sensing region.Attorney Docket No. 206085-0196-00WO

[0152] 14. The method of embodiment 13, wherein the method comprises applying a high-frequency signal to the SRR and obtaining the change in capacitance at the gap in the inner ring to measure the electrical diameter of the at least one cell, and applying a low-frequency signal to the Coulter counter and obtaining the change in current across the electrodes to measure the geometric diameter of the at least one cell.

[0013] 15. The method of embodiment 14, wherein the method comprises comparing the geometric diameter and electrical diameter of the at least one cell exposed to the drug with a baseline measurement.

[0154] 16. The method of embodiment 15, wherein the baseline measurement is the geometric diameter and electrical diameter of the at least one cell without being exposed to the drug.

[0155] 17. The method of embodiment 15, wherein the cell is determined to be susceptible to the given dose of the drug when the geometric diameter is increased or decreased by at least 5%.

[0156] 18. The method of embodiment 15, wherein the cell is determined to be susceptible to the given dose of the drug when the electrical diameter is increased or decreased by at least 5%.

[0157] 19. The method of embodiment 11, wherein the cell is a cancer cell.

[0158] 20. The method of embodiment 19, wherein the cell is a tumor cell.

[0159] 21. The method of embodiment 11, wherein the drug is a cytostatic, cytotoxic, or mixed-effect drug.

[0160] 22. A method of treating cancer in a subject comprising determining that a population of cancer cells from the subject is susceptible to a drug by the method of embodiment 10, and administering the drug to the subject.EXPERIMENTAL EXAMPLES

[0161] The invention is now described with reference to the following Example. These Examples are provided for the purpose of illustration only and the invention should in no way beAttorney Docket No. 206085-0196-00WOconstrued as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0162] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore specifically point out exemplary embodiments of the present invention and are not to be construed as limiting in any way the remainder of the disclosure.Example 1 : Single-Cell Microwave Cytometry for Drug Resistance Detection in Cancer

[0163] An emerging new approach is functional precision medicine where the effectiveness of specific drugs are directly tested on living cells isolated from patient biopsies and their efficacy immediately evaluated using in vitro standard viability assays (Flaherty et al., 2020, Journal of Clinical Oncology. 38:3883-3894; Friedman et al., 2015, Nature Reviews Cancer. 15:747-756). However, functional precision medicine encountered limitations in wide-scale use due to large sample requirements, long turn-around times, genetic drift in isolated samples, and the loss of cell viability ex vivo (Popova and Levkin et al., 2020, Advanced Therapeutics.3:1900100). The challenges mentioned above can be overcome by the advent of rapid microfluidic-based single-cell biosensors.

[0164] In the field of single-cell sensors, monitoring changes in cellular biomass induced by drug exposure emerged as a robust and non-invasive assay for functional precision medicine in recent years (Kimmerling et al., 2024, Cancer Research. 84:5177; Stevens et al., 2024, JCO Precision Oncology. 8:e2300249). Cellular mass measurements probe cellular composition which is affected by the action of drugs. Cellular mass is measured most sensitively by suspended microchannel resonators (SMRs), where a hollow micro-cantilever with an integrated microfluidic channel carries the media containing single cells (Burg et al., 2007, Nature.446:1066-1069; Stevens et al., 2016, Nature Biotechnology. 34:1161-1167; Cermak et al., 2016, Nature Biotechnology. 34:1052-1059). This assembly is oscillated in vacuum to obtain the buoyant mass of each cell passing through the hollow micro-cantilever. In certain cases, SMR technology can detect cell pathology by means of measuring changes in cellular mass even more rapidly than through conventional assays (Kimmerling et al., 2022, Communications Biology.Attorney Docket No. 206085-0196-00WO5:1295). This observation, in combination with high-throughput microfluidic-based measurements and low sample volumes allows the development of novel testing modalities with faster turnaround times for informed drug selection. However, focusing on cellular changes solely in one dimension such as cellular mass — which significantly varies through the cell cycle — coupled with the delicate device and apparatus requirements to accomplish such measurements has so far limited the reach and scope of this approach. Recent advances have highlighted the potential of multi-parametric biophysical measurements for cell density, including high-throughput profiling using fluorescence exclusion microscopy coupled with SMRs (Wu et al., 2025, Nature Biomedical Engineering. 1-10). Here, a different approach is taken to probe cellular composition electronically and along multiple dimensions by using a simple microfluidics system which can be fabricated in standard cleanroom facilities. With this system, it is possible to simultaneously obtain cellular volume, electric size (an analogue of dry mass in the electrical domain), and dielectric permittivity. These parameters can then predict the outcome of an anti-cancer drug treatment.

[0165] Recently, the development of an electronic sensor was reported which integrates a low-frequency Coulter counter, and a high frequency microwave resonator to probe single microparticles such as microplastics and single cells (Tefek et al., 2023, Advanced Materials. 35:2304072). These sensors simultaneously measure the cellular volume and electrical size. Electrical size measurements operate at microwave frequencies, which bypass ionic contributions and instead detect the dielectric contrast between cellular biomaterial and water — making electrical size an electronic analogue of a cell’s dry mass. By combining the cellular volume and electrical size, it is possible to obtain the microwave permittivity of cells in a high-throughput manner. In this sense, the measurements in the microwave domain yield a metric for the amount of biomaterials and water inside cells, thus providing a unique fingerprint for the material composition of a cell. When the frequency of measurement lies within the dipolar polarization regime (1-10 GHz), microwave measurements yield the electrical analogue of cellular mass and mass density, which can be utilized to monitor the biophysical state of a cell when exposed to drugs (Figure 1).

[0166] Indeed, the changes in the biomaterial-to-water ratio inside a cell are better reflected in the microwave permittivity of the cell, rather than the mass density. This is because theAttorney Docket No. 206085-0196-00WOdifference in electrical polarizability is very large between water (c_r~70) and hydrocarbon chains inside organic groups (e_r=2-3); on the other hand, there is not much difference between these two types of components in terms of mass density (1.0 g / cm3vs 1.4 g / cm3). Thus, the internal permittivity of a cell is inherently a more sensitive probe into the composition of a cell. Owing to the simplicity of the sensor fabrication, the absence of any high frequency mechanical oscillations and suspended structures with narrow channels, and independence from optical or biochemical labelling, the sensor avoids the pitfalls of state-of-the-art technologies in terms of complexity, high-cost, and the need for expert operators, while maintaining all the benefits of high-throughput microfluidic-based sensors, offering flexibility, sensitivity, and speed.Moreover, the developed platform requires only small sample sizes on the order of a few thousand cells per measurement set.

[0167] Here, the capabilities of this sensor are demonstrated by first investigating changes in volume and electrical size of single cells by doing a proof-of-principle experiment where hypo-and hypertonic solutions were used to induce water uptake in human embryonic kidney (HEK293 FT) cells. By combining these measurements, the changes in cell microwave permittivity (a) were tracked as a function of solution osmolarity at the single-cell level. For drug testing, a human colorectal carcinoma model (DLD-1) was used to monitor the effects induced by gefitinib (EGFR inhibitor) on both parental and drug-resistant variants using measurements of geometric and electrical size, permittivity, and — for independent verification — Trypan Blue viability assay. The same cell populations were then exposed to a different drug (cisplatin) to verify whether the observed resistance was drug-specific and whether single-cell microwave cytometry can resolve the drug-specific effects. These measurements were extended for two more cell lines (in each case with parental and resistant variants) against cisplatin and tamoxifen. These three drugs represent different modes of action of cancer therapeutics — relatively cystostatic (gefitinib), cytotoxic (cisplatin) and mixed-effect i.e., both cytostatic and cytotoxic (tamoxifen) — allowing us to evaluate sensor’s ability to distinguish diverse biophysical signatures associated with distinct modes of action. Indeed, in all cases, the electronic measurements recapitulated the biological observations about the cellular viability, drug susceptibility, and development of drug resistance accurately. Finally, the sensor was tested with Patient-Derived Organoid (PDO) models established from metastatic colorectal carcinoma from a cancer patient against two drugs with known differential efficacy — bortezomib (effective) andAttorney Docket No. 206085-0196-00WOgefitinib (ineffective). Overall, the results demonstrate that cellular drug response can be assessed accurately by this integrated electronic approach through single-cell measurements and at high throughputs, underscoring its potential as a functional precision medicine tool for guiding therapy selection.Results

[0168] Probing the effects of hypo- and hypertonicity on cell volume and permittivity:Measuring the microwave permittivity (e) of the cell interior provides an effective assessment of the ratio between biomaterial and water content of the cell. Biomaterial content encompasses proteins, nucleic acids, carbohydrates, and lipids. The relative permittivity of pure water is approximately 70 at microwave frequencies, whereas proteins exhibit relative permittivity values ranging from 2.5 to 3.5 (bulk values (Amin & Kiipper, 2020, Chemistry Open. 9:691-694)), resulting in a substantial and measurable contrast between these materials. Additionally, the biomaterial-to-water ratio dictates the reaction kinetics inside the cell, by determining the reaction rates and equilibrium concentrations in biochemical processes (Neurohr & Amon, 2020, Trends in Cell Biology. 30:213-255). The biomaterial-to-water ratio exhibits itself in cell mass density which is a tightly regulated property of mammalian cells, (Neurohr & Amon, 2020, Trends in Cell Biology. 30:213-255; Grover et al., 2011, PNAS, 108:10992-10996; Durmus et al., 2015, PNAS. 112”E3661-E3668) as variations in enzyme concentration shift the biochemical equilibrium and can affect proper biochemical functions, structural integrity, and protein folding (van Tartwijk & Kaminski, 2022, Advanced Biology. 6:2101328; Chen et al., 2024, Nature Communications. 15:2149). Notably, only a small number of processes can affect cell mass density, including cell division, differentiation, apoptosis, senescence, and changes in extracellular osmolarity (Neurohr & Amon, 2020, Trends in Cell Biology. 30:213-255). On the other hand, protein synthesis and protein degradation appear to have minimal impact on cell density (Li et al., 2022, Frontiers in Cell and Developmental Biology. 10:1017499).

[0169] Under hypotonic stress, cells uptake water to equalize osmotic pressure across the lipid membrane. This uptake naturally entails a change in the intracellular density. At osmolarities as low as -140 mOsm / L (i.e., approximately half the physiological osmolarity), cells generally maintain their nominal volume (Bozic et al., 2020, Frontiers in Physiology.11 :582781), and only begin to uptake water at smaller osmolarities. To assess whether theseAttorney Docket No. 206085-0196-00WOchanges in density can be measured by our sensor, HEK293 FT cells were subjected to various concentrations of PBS solution at varying concentrations (between 10% and 150%) and the cell volume and electrical size were measured, at the microwave band, approximately one minute after resuspension. Fig. 2a illustrates the changes in the geometric and electrical volume of HEK293 FT cells at varying concentrations (10%, 20%, 50%, 100%, and 150%; 28.8, 57.5 ,143.8, 287.5, and 431.2 mOsm / L, respectively). Microwave permittivity (s) was derived by normalizing the electrical diameter to the geometric diameter. Fig. 2b illustrates the changes in permittivity (left-axis) and geometric diameter (right-axis) by Earth Mover’s Distance (EMD) which is a measure of dissimilarity between two statistical distributions (Orlova et al., 2015, PLoS One. 11 :e0151859). An interesting effect was observed in geometric volume for diluted concentrations: moving from an isotonic condition to 50% dilution, the change in the geometric diameter was quite small (<10% increase) matching what has been shown in the literature (Bozic et al., 2020, Frontiers in Physiology. 11 : 582781). However the geometric diameter changed significantly for the 20% and 10% dilutions (+25% and +38% increase respectively).

[0170] Concurrently, we also observed a change in the electrical permittivity (e) matching the expansion in cell volume. The change was minimal at 50% dilution (~1% increase) resulting from the small volumetric increase and uptake of water. This change rose to 3% and 4% for the 20% and 10% dilutions respectively. Normally under hypotonic conditions, cells facilitate the efflux of electrolytes (Na+, K+, and C1-) to equilibrate the cytosolic osmolarity with the external environment. However, at high (GHz) frequencies, ions in solution do not significantly contribute to polarization (and thus s) extracted by our sensor, because the ionic current is significantly dampened at GHz frequencies (Hwang, 2021, IEEE Microwave Magazine. 22:78-87). Therefore, it was hypothesized that the change in e is due to compositional changes relating to the ratio of biomaterial in the cell, relative to water, starting to decrease with water uptake, as opposed to the ionic concentration decreasing in the cytosol. Previous studies have shown that cells indeed expel organic osmolytes in addition to electrolytes in the early stages of exposure to a hypotonic environment in order to equilibrate osmotic pressure before resorting to volumetric changes (Bozic et al., 2020, Frontiers in Physiology. 11:582781; Groulx et al., 2006, The Journal of Membrane Biology. 214:43-56). In contrast, at more extreme dilutions, the cells will finally resort to water uptake in order to equalize pressure as seen at 20% and 10% PBS concentrations, but the trend of increasing s continued as the cell mass density decreased in parallel.Attorney Docket No. 206085-0196-00WOFurthermore, using normalized Raman imaging, Liu et al. (2022, Frontiers in Cell and Developmental Biology. 10:1017499) reported that the dry mass of cells is affected quite noticeably by external osmotic stress, where cells grown in hypotonic solutions demonstrated considerably less dry mass than cells grown in isosmotic solutions. This correlates well with the permittivity increase that we detected as a function of decreasing PBS concentration.Consequently, it is reasonable to deduce that the compositional change indicated by the change in s at microwave frequencies is largely independent of ionic concentration, conductivity, or ionic polarizability, and is instead predominantly dependent on the total polarizability of the intracellular biomaterials relative to water. As the biomaterial-to-water ratio decreases due to water uptake, the permittivity of the cell interior increases. This change in the permittivity signal was further confirmed by analysing cells in a more concentrated (hypertonic) PBS solution. At 150% of the original concentration, we observed a significant decrease in cell geometric size, down by -10%, accompanied by a corresponding decrease in relative permittivity of -3%. This change in size was due to water expulsion, while the decrease in permittivity reflects the internal biomass-to-water ratio increase due to the loss of water through dehydration.

[0171] Conducting single-cell measurements at the microwave band provides a novel mode of probing the internal composition of cells, distinct from impedance cytometry. Here, compositional changes detected by the sensor are analogous to the mass density of a cell, which in this context would be indicated by an 'electrical density' value. Given that the biomaterial-to-water ratio, or cell density, is a tightly controlled parameter, significant alterations in the measured s value can indicate pathological conditions within a cell population or substantial compositional changes due to extraordinary environmental conditions experienced by the cell population.

[0172] Drug resistance probed by microwave cytometry: It was previously established (Tefek et al., 2023, Advanced Materials. 35:2304072) that the change in cell permittivity at microwave frequencies is a strong indicator of compositional changes inside a cell following the application of a fixation agent. Compositional alterations are commonly observed in cancer cells as well, such as nuclear enlargement leading to an increase in nuclear-to-cytoplasm ratio, as well as hyperchromasia which increases nuclear density (Fischer, 2020, Cytologica. 64:511-519; Zink et al., 2004, Nature Reviews Cancer, 4:677-678). Although solid tumors are stiff, individualAttorney Docket No. 206085-0196-00WOcancer cells are softer than non-tumor cells, due to re-arrangement of actin / cytoskeleton networks to allow for flexibility to metastasize (Alibert et al., 2017, Biology of the Cell.109: 167-189). Such changes in the cytoskeleton are expected to shift the electrical polarizability of cells.

[0173] The intracellular structure of cancer cells also changes in response to the presence of physiological stressors such as drugs. In fact, Kimmerling et al. were able to show a clear link between drug exposure and changes in the distribution of cellular mass using a SMR mass sensor (Kimmerling et al., 2022, Communications Biology. 5:1295). The changes in cellular mass were observed after exposure to over 60 different types of drugs spanning various mechanisms of action over twelve different cancer types. Instead of inferring compositional change inside the cell using a single parameter (buoyant mass), the present sensor provides multi-dimensional information about the cell: electrical size (an analogue of dry mass), geometric size (obtained electronically using the same sensor), and microwave permittivity. This capability allows us to monitor internal changes in the cellular structure and content accurately to determine whether a specific cancer cell line is resistant to a specific drug or not. Isogenic pairs of parental and resistant cell line models were used to ensure that the difference between sensitive and resistant cells are only due to the drug being investigated. This is critical for our approach as we need to test our platform’s capacity to detect sensitivity and resistance with high precision and dynamic range upon drug treatment.

[0174] Here, in the first set of experiments, the colorectal cancer DLD-1 cell line was used as the parental (or sensitive) for a series of drug testing experiments. Starting with parental DLD-1 cells (DLD1.Par), an isogenic cell line DLD1 GefR was generated which acquired resistance to gefitinib in vitro through prolonged exposure to the drug (Methods and Supplementary Information sections) (McDermott et al., 2014, Frontiers in Oncology. 4:40; Uramoto et al., 2006, International Journal of Clinical Oncology. 11:487-491; Zhang et al., 2008, Oncology Reports. 19:1541-1544).

[0175] Following the acquisition of gefitinib-resistant populations, both parental and gefitinib-resistant populations were incubated in growth media spiked with increasing concentrations of gefitinib (0, 10, 20, 30, 40, and 50 pM) for 72 h. Figure 3 describes the measurement results of the cell populations for both geometric size and electric size asAttorney Docket No. 206085-0196-00WOpercentage changes from nominal values for cells in ideal growth conditions using our sensor. Cell viability was measured in parallel (Methods section). The effects of the drug on cell populations can be followed in a two-dimensional plane for geometric and electric size change for cells (Figure 3a). In this representation, the changes in cellular volume such as swelling or shrinkage appear as changes along the x-axis, while the compositional changes in the electrical domain have a projection along the y-axis where higher values represent a higher intracellular biomaterial content and vice versa. For parental (gefitinib-sensitive) cells, even at the 10 pM concentration, we observe a significant swelling in cell geometric size, accompanied by a slight increase in electrical size (Figure 3a, circle markers). This might have resulted from increased water uptake as an adaptation to the drug by the cells. Even at these small drug concentrations (10 and 20 pM), the cell viability (seen as colormap-coded symbols in Figure 3a) appeared to decrease dramatically (-50%).

[0176] The viability continued to decrease with increasing gefitinib concentrations, reaching 10% at the highest concentration of drug (50 pM). Concurrently, at concentrations >20 pM, the cells maintained their swelling, but the electrical size increased rapidly as well, indicating an increase in cell density. This might be a consequence of cellular hypertrophy (Hbpfner, 2003, British Journal of Cancer. 89:1766-1775; Blagosklonny, 2006, Journal of Cellular Physiology.209:592-597; Crozier et al., 2022, bioRxiv. 2022.2009.2008.506843; Demidenko et al., 2009, Cell Cycle. 8:1888-1895). By contrast, this trend was not observed in gefitinib-resistant cells (Figure 3a, diamond markers). In fact, both geometric size and permittivity remained nearly unchanged until concentration values exceeded 30 pM. For concentrations over the 30 pM threshold, a slight decrease in size was observed along with a slight increase in electrical size, which might indicate a loss of water content when coupled with the decrease in size. This is a significant observation, since gefitinib-resistant cells were kept in 30 pM gefitinib media prior to performing the experiment to maintain their tolerance to the drug, so it would be reasonable to assume that the cells will only begin to experience negative effects at concentrations greater than 30 pM. This is further supported by viability measurements where we observe an increase in cell death (defined as 100% - cell viability) from approximately 5% to 20% transitioning from 30 to 40 pM concentration (Figure 3b). Generally, it seems that cell populations that experience more negative effects from drugs tend to exhibit more severe structural changes either in geometric size or in density (electrical size). Therefore, it is useful to look at the extent of the overallAttorney Docket No. 206085-0196-00WOchange in both dimensions as an indicator of drug susceptibility. Overlaid with the viability measurements in Figure 3b, we also plot the total EMD distance (norm-2 distance of electric and geometric EMD values). The total sensor response and the cell viability measurements run in parallel to each other in this case (Figure 3b) indicating the connection between the electronic measurements and the viability of the cells.

[0177] Cisplatin response in gefitinib-resistant cells: The results above showed that our sensor system can predict the response of gefitinib-resistant and parental cells under gefitinib treatment by tracking the structural change they experience relative to nominal values in the control samples. Next, whether a different drug (cisplatin) would have a noticeable effect on gefitinib-resistant cells was investigated. Given that the gefitinib-resistant cell line was not necessarily resistant to other drug classes, the exposure to cisplatin (a cytotoxic DNA-damaging drug) is expected to yield dramatic changes in the geometric and electrical size of gefitinib-resistant cell line.

[0178] First, the gefitinib-resistant cell line (DLD1 GefR) was exposed to 0, 10, 20, 30, 40, and 50 pM of cisplatin as described in the Methods section. The sensor response for the DLDl.GefR cell line was compared with respect to the application of gefitinib vs. cisplatin in Figure 4a. In this case, DLDl.GefR cells demonstrated sensitivity towards cisplatin where at the smallest dose (10 pM) as observed by cell death. Indeed, at this dose, a marked increase in the sensor reading (EMD Total Response) was observed compared to the case with gefitinib. As the concentration is increased, the cisplatin-treated cells keep producing larger response in the sensor compared to their gefitinib-treated counterparts, and in parallel with the cell viability measurements superimposed on the graph.

[0179] Figure 4b compares the response of DLD1.GefR vs. DLD1.Par under the effect of cisplatin. In this figure, the electrical and geometrical responses of DLDl.GefR vs. DLDl.Par cell lines are plotted as a function of cisplatin concentration, superimposed on the results from the previous two experiments (with gefitinib) for comparison. For the gefitinib-resistant cell line (triangles, DLD1.GefR), an increase in geometric size (swelling) and an increase in electric volume (the analogue of dry mass) are seen. This was followed by a sharp decrease in geometric size for intermediate drug concentrations (20-30 pM) with a small decrease in electrical size, which could be consistent with an apoptotic trajectory where a cell loses between 20-40% of itsAttorney Docket No. 206085-0196-00WOoriginal volume, (Lopez-Hernandez, 2021, Cell Physiol Biochem. 55:161-170) followed by membrane blebbing and the expulsion of intracellular components (Saraste & Pulkki, 2000, Cardiovascular Research. 45:528-537). Finally, the changes in cellular volume and electrical size saturate at the highest dose levels where both the size and density of cells are lower than nominal cell values in the control sample. Remarkably, the use of a cytotoxic drug (cisplatin) and cytostatic drug (gefitinib) charts different trajectories in this plane: for instance, cisplatin causes the cells to die as they shrink consistent with an apoptotic trajectory. On the other hand, gefitinib-treated cells die at larger volumes, indicating the result of cell cycle arrest and hypertrophy consistent with the cytostatic nature of the gefitinib.

[0180] Parental DLD-1 cell lines (DLD1.Par) exhibited a smaller response, compared to the gefitinib-resistant cell lines (Figure 4b blue trace, square symbols) indicating better tolerance to cisplatin. The tolerance of DLD1. IPar can be understood in the light of a previous study by Fernandez et al, (2008, Molecular Cancer Therapeutics. 7:327-3246) which showed that DLDl.Par cells moderately tolerated ~40 pM of cisplatin exposure, with 35% of cells remaining viable after 96 h of treatment. Using the aforementioned study as a benchmark, our viability results (Figure 8) showed that parental DLDl.Par cells exhibited a 62.9% viability following incubation with 40 pM cisplatin, and at 50 pM cisplatin concentration the viability dropped to 48% after 72 h of drug exposure. This indicates that the DLDl.Par cells tolerated cisplatin at the concentration range applied here. Interestingly, both electrical and geometric size hardly changed across the same concentration range (Figure 4b, square markers). The seemingly unexpected observation where the gefitinib-resistant cell line was more sensitive to cisplatin than the parental cell line could be explained by the possibility of a synergistic effect between gefitinib and cisplatin as detailed in Figure 8.

[0181] The seemingly unexpected gefitinib-resistant cell line sensitivity to cisplatin (Figures 4B and 8) could be explained by the possibility of a synergistic effect between gefitinib and cisplatin. Previous studies have shown that EGFR-TKIs (such as lapatinib) chemosensitize ovarian cancer cells to cisplatin (Coley et al., 2006, Biochemical pharmacology. 72:941-948), while EGFR-inhibitor osimertinib combined with pemetrexed or cisplatin showed effective tumor suppression in lung cancer (La Monica et al., 2019, Journal of Experimental & Clinical Cancer Research. 38:1-12). Ahsan et al. (2010, Cancer research. 70:2862-2869) showed thatAttorney Docket No. 206085-0196-00WOcisplatin induced EGFR phosphorylation and degradation in wild type human head and neck carcinoma (Ahsan et al., 2010, Cancer research. 70:2862-2869). Furthermore, they also showed that this degradation effect was enhanced by the administration of EGF, thus cells that rely on that receptor for growth (which is the case with gefitinib-resistant DLD-1) might be more susceptible to EGFR degradation through cisplatin-induced cytotoxicity. It is also worth noting that a randomized phase III study, concluded in 2017, evaluated pemetrexed / cisplatin as a first-line therapy for patients with non-small cell lung cancer tumours with EGFR-activating mutations (NCT00949650; Ahsan et al., 2010, Cancer research. 70:2862-2869). This leads to the conclusion that the enhanced sensitivity to cisplatin exhibited by gefitinib-resistant cells might be due to EGFR degradation. In this specific case, both geometric size and permittivity plummeted at the first administered cisplatin dose, and continued to fall further, possibly indicating an apoptotic trajectory for death, highlighted by a rapid loss of size and the condensation of the biomaterial content.

[0182] The results thus far suggest that DLD-1 parental cells would undergo drastic compositional changes both geometrically and electrically when exposed to a drug they were sensitive to. This trend is in line with similar but diminished changes observed in the resistant lines.

[0183] Broad applicability across drugs with diverse mechanisms of action. Since cells differ significantly in their response to drugs, it was determined whether these compositional changes were a universal trait when cells are exposed to drugs that they were sensitive to. To this end, earlier results on a cytostatic drug (gefitinib) was complimented with cytotoxic (cisplatin) and mixed-effect (tamoxifen) drugs, and corresponding isogenic cell lines. For cisplatin the parental HCC-1937.Par and its cisplatin-resistant variant HCC-1937.cisR were used. For tamoxifen, the MCF-7.Par parental cell line and its tamoxifen-resistant variant MCF-7.tamR were used. The concentration range of each drug was adjusted based on the IC50 concentrations determined for the parental cell lines against each drug (Figures 13-15). Given the observation that both geometric and electric sizes exhibited significant changes under the stress of drugs, the cumulative shifts in both dimensions was quantified using the Hotelling T2 test which is a measure of the difference between two statistical distributions in two dimensions. Hotelling T2 scores provide a single value that is commensurate with changes in both of those dimensionsAttorney Docket No. 206085-0196-00WOrelative to the origin point (control sample). The results for the Hotelling T2 for each drug / pair combination are shown in Figure 5 including the previously tested DLD1 isogenic cell lines to allow comparison. In Figure 5, a large value of Hotelling T2 (bar plots) indicates a large change in the electrical and geometric diameters of the cell line under the influence of the applied dose of the drug. As visible from Figure 5, in all cases, there is a large Hotelling T2 peak for the parental (drug sensitive) cell lines which indicates a sudden change in biophysical parameters at the applied drug level. Indeed, the Hotelling T2 peaks emerge near concentration values where cellular viability has a sharp drop (which in turn are often close to the LC50 values). These results on different cancer types further indicate the utility of our technique for predicting the response of the drug.

[0184] Examining population distributions in both geometric and electrical dimensions offers detailed insights into the evolution of cell populations under drug influence. This is visualized in the violin plots for each drug / cell pair (Figures 5B, 5D, and 5F). Key indicators include abrupt shifts in population distribution peaks and changes in population dispersity or broadening.

[0185] In the gefitinib vs. DLD-1 violin plot (Figure 5B), no significant changes in geometric or electrical diameters were observed, except for the parental cell population within the 0-10 pM range, which corresponds to a pronounced Hotelling peak. Another notable feature is the widening of the electrical diameter peak for the parental population at 30 pM, mirrored by a similar widening in the resistant population later on at 50 pM.

[0186] Given gefitinib ’s cytostatic nature, more pronounced compositional changes in cell populations exposed to a cytotoxic drug like cisplatin was expected. Indeed, the violin plots for DLD-1. GefR vs cisplatin (Figures 9 and 10) do show drastic geometric shifts and extensive peak broadening starting at the 10 pM concentration. These shifts are much more drastic than DLD-l.Par vs gefitinib. We also see peak broadening in the electrical domain for DLD-1. GefR vs cisplatin starting from the lowest concentration, compared to minimal peak broadening in the electrical domain for DLD-1.Par vs gefitinib except at concentrations of 30 pM and larger.

[0187] Similarly, the application of the cisplatin on HCC-1937 indicates dramatic reductions in both geometric and electrical diameters are evident for parental cells at 25 pM drug concentration (violin plot in Figure 5D). These drops are preceded by gradual increases in size atAttorney Docket No. 206085-0196-00WOlower drug concentrations, with the highest Hotelling peak occurring at 25 pM for the parental line (Figure 5C). In contrast, resistant cells exhibit no such sharp decreases; however, significant increases in dispersity suggest a heterogeneous response within the resistant population, where some cells experience adverse effects while others tolerate the drug better.

[0188] For the application of tamoxifen on MCF-7 cells, we see that at low dose levels (5 pM and 10 pM), the MCF-7 Par cell line undergoes a quick loss of viability, dropping below 40% (Figure 5E). Within this range, the viability of the MCF-7. TamR stays at relatively higher levels. These observations are recapitulated in the sensor response curves, as the MCF-7. Par response value is almost double, compared that of MCF-7. TamR. At the highest concentration levels (>15 pM), viabilities converge to 0 % for both types (i.e. all cells die); as such, these dramatic drops in both types exhibit very large sensor responses. This is reminiscent of the earlier case with HCC-1937.Par response at 25 pM concentration of cisplatin (Figure 5C), where the full extinction of cells was accompanied by a large change in sensor response. The geometric and electrical diameter distributions for the MCF-7. Par and MCF-7. TamR cell lines are provided in Figure 5F.

[0189] The distribution patterns of electrical and geometric diameter reveal a key distinction: resistant cells tend to maintain the overall geometric and electrical size under drug exposure except when the drug dose is too high, while parental cells display rapid deformations, such as sudden changes in geometric and electrical size, coupled with distribution broadening or splitting into sub-populations. Notably, resistant cells also exhibit distribution broadening and population splitting especially at higher dose levels, which might indicate a heterogeneous response to the drug among the resistant population, especially at higher drug doses.

[0190] These findings provide valuable insights into the mechanistic effects of specific drugs on various cell lines, enabling a deeper understanding of drug susceptibility and resistance in terms of their effect on cellular composition.

[0191] Measurements on patient-derived organoids: To further generalize our observations, the same technique was applied on patient-derived organoids (PDOs), highlighting its potential for translational applications (Zhang et al., 2022, Trends in Biotechnology. 40: 1121-1135). PDOs provide a model system that closely mimics the heterogeneity of tumors (Drost &Attorney Docket No. 206085-0196-00WOClevers, 2018, Nature Reviews Cancer. 18:407-418). By using cells directly from patients, this approach demonstrates an accurate investigation of their therapeutic response (Vlachogiannis et al., 2018, Science. 359:920-926) . Generally, primary cells grown in 3D organoid cultures are expected to recapitulate the response in vivo more faithfully than conventional 2D cell cultures owing to their growth conditions and geometry (Suarez-Martinez et al., 2022, Cell&Bioscience.12:39). As a result, the demonstration of microwave cytometry on PDOs serves as an important proof-of-concept towards clinical applicability.

[0192] The effects of Gefitinib and Bortezomib on the cell viability of the PDOs from the same donor were previously assessed using the Sytox-Green nucleic acid staining procedure, followed by 3D nuclear visualization for quantitative analysis (manuscript in preparation).Treatment with 1 pM Gefitinib and 1 pM Bortezomib resulted in cell viabilities of -100% and <10%, respectively, compared to the control group treated with DMSO. Consequently, three organoid groups from the same donor were grown in optimal conditions (Methods) and two of those groups were then incubated with gefitinib and bortezomib respectively at concentrations of 1 pM, while the third was maintained in optimal conditions and served as control. Following the growth and incubation periods, the organoids were dissociated into single cells through gentle digestion using TrypLETM and then passed through the sensor.

[0193] The analysis revealed distinct differences in the cell populations. While the control population and the gefitinib-treated population aligned closely along the 2D geometric versus electric diameters plot (Figure 6A), cells treated with bortezomib were noticeably smaller in size. A clear trend emerged: gefitinib-treated cells were slightly smaller in both geometric and electric diameters compared to the control, while bortezomib -treated cells were significantly smaller in both parameters.

[0194] Statistical analysis using the Hotelling T2test confirmed significant variation in both geometric and electric diameters for the bortezomib-treated population compared to the control (Figure 6B). In contrast, the gefitinib-treated population showed only minor variations relative to the control group. Observing the data at finer granularity, the violin plot (Figure 6C) shows that the gefitinib-treated sample shows two sub populations where possibly some individual cells tolerated the drug better than their counterparts in the other sub population. This becomes clearer when compared to the bortezomib-treated population where a single population appeared with aAttorney Docket No. 206085-0196-00WOpeak value that deviated sharply from the control sample, indicating that almost all the cells in the population experienced adverse effects due to their exposure to the drug.Conclusions

[0195] This work shows for the first time that the combination of microwave and low-frequency electronic measurements could be used to determine the susceptibility of a cell line to a given drug. First, the sensor’s ability to measure changes in the biomaterial-to-water content of cells was assayed by exposing them to salt solutions with different osmolarities. A drug susceptibility study was then conducted using a pair of DLD-1 cell lines — one of which was sensitive and the other resistant to gefitinib. The effects of gefitinib on these cell lines were successfully monitored across multiple biophysical dimensions. The parental cell line exhibited swelling and significant alterations in microwave permittivity which is indicative of changing biomaterial-to-water ratio. In contrast, the resistant cells showed almost no change in sensor response, even at concentration up to 30 pM — the level at which they were incubated to maintain drug resistance. By contrast, administering a different drug, cisplatin, to the gefitinib-resistant cell line had an immediate effect in the sensor response.

[0196] Subsequently, this methodology as applied to additional isogenic cell lines and associated drugs to demonstrate applicability in the major drug mechanisms: cytostatic (gefitinib), cytotoxic (cisplatin) and mixed-effect (tamoxifen) drugs. In each case that the electronic measurements could distinguish between the sensitive and resistant cells. In all cases, the response of the sensor was validated through cell viability measurements. Moreover, the ability to disentangle volumetric and compositional changes, and the analysis of the effects of cytostatic and cytotoxic drugs on cells tested here, indicate the potential of microwave cytometry for identifying mechanisms of actions for different drugs.

[0197] Finally the potential for translational research and clinical application was shown by analyzing cells from PDOs. These findings demonstrate that this approach can provide important insights into cell viability of primary cells in PDOs better reflecting tumor heterogeneity, offering a potential clinical application. Other biological samples, such as blood, pleural effusion and fine-needle aspirates can also be used in our system after isolation of cancer cells but withoutAttorney Docket No. 206085-0196-00WOthe need for culturing, since our microfluidics platform and single-cell technology can provide drug-resistance information by using only several thousand cells.

[0198] Given the versatility of this system in testing diverse drugs on single cells from both cultured cells and PDOs, this approach offers a powerful avenue to tailor patient-specific treatment modalities, advancing precision medicine and ultimately improving clinical outcomes.Materials and Methods

[0199] Sensor device fabrication: The sensor device was comprised of a planar split-ring resonator (SRR) design deposited on fused silica wafers (Quartz Unlimited LLC). Device fabrication has been described in detail in previous publications (Tefek et al., 2023, Advanced Materials. 35:2304072; Secme et al., 2023, IEEE Sensors Journal. 23:6517-6529). Briefly, standard soft lithography was utilized to deposit a thin Au layer to form the body of the SRR using a pre-etched Cr photolithography mask. The SRR comprised of two concentric rings, one larger outer ring, and another smaller inner ring. The outer ring was split, with two Au traces coming out of each end and terminating with a sub-miniature A (SMA) connector soldered onto the edge of the fused-silica wafer. The inner ring comprised of another split circle where the split comprised of a small region where signal sensing took place. The sensing region in the inner ring consisted of two asymmetrical gaps spanning a length of approximately 250 pm across (Figure 7). Furthermore, two Au electrodes were deposited surrounding the SRR sensing region and extending outwards for subsequent wire bonding. These electrodes were connected to a low frequency signal generator and acted as a conventional Coulter counter. Finally, a straight (100 pm wide, 45 pm tall, and 70 mm long) microfluidic channel with a constriction (40 pm wide, 45 pm tall, 250 pm long) in the center was developed in polydimethylsiloxane (PDMS, Dow Chemicals) featuring an inlet and outlet at either ends. This channel was then carefully aligned and bonded (through O2 plasma treatment) so that the constriction in the channel covered the sensing region of the SRR precisely.

[0200] Liquid containing samples was delivered through the microfluidic channel using a Fluigent (MFCS-EZ) pressure control system. Typical flow rates ranged from 8 to 15 pL / min with a cell transit time through the SRR sensing region being between 10-20 ms.Attorney Docket No. 206085-0196-00WO

[0201] Electronic measurements: The sensor platform consists of a low-frequency and a high-frequency (microwave) sensor to obtain the internal property of the cells. In the low-frequency part, referred as Coulter counter, an insulating particle’s passage results in partial blockage of ionic current being conducted between two electrodes, the decrease in current being proportional to the geometrical volume of the particle. A 0.5 VPPsignal at 0.5 MHz from a lock-in amplifier (Zurich Instruments, HF2LI) was used to drive one electrode while the resulting current flowing through the ionic solution was collected from the other electrode. This signal was converted to voltage by a transimpedance amplifier (Zurich Instruments, HF2TA) and consequently read out by the lock-in amplifier.

[0202] The high-frequency part of the sensor utilized a split ring resonator (SRR) for electrical size measurement of the cells. The SRR contains two concentric rings, the inner ring is excited inductively by the microwave signal fed through the outer ring. A highly concentrated electric field is created in the gap (split region) of the inner ring due to the standing wave mode (Abduljabar et al., 2014, IEEE Transactions on Microwave Theory and Techniques. 62:679-688; Lee & Yook, 2008, Applied Physics Letters. 92; Salim & Lim et al., 2016, Sensors. 16:1802) and passage of particle through the gap cause phase and amplitude change of the resonator due to capacitance change in the sensing region representative of the particle’s geometric volume and the permittivity. In this specific design, time delay of the signals from two gaps of unequal width (15 and 25 pm) in the inner ring were utilized to obtain the height information of the passing particles and for calibrating the signal (Figure 7).

[0203] A vector network analyzer (VNA) was used to observe resonance characteristics of the SRR before connecting to the custom measurement circuitry for conducting measurements (Tefek et al., 2023, Advanced Materials. 35:2304072). A signal generator was used as a signal source and its frequency was set as the resonance frequency of the SRR (=5.3 GHz). A 0.5 VPPsignal at the resonance frequency was fed to the SRR. Two lock-in amplifiers (Zurich Instruments, MFLI) were used to employ single side band modulation (SSBM) as part of the measurement circuitry. A custom-built single side band (SSB) heterodyne circuitry (Ferrier et al, 2009, Lab on a Chip. 9:3406-3412; Nikolic-Iaric et al., 2009, Biomicrofluidics. 3; Kelleci et al., 2018, Lab on a Chip. 18:463-472) was used as the resonance frequency was higher than the maximum operational frequency of the lock-in amplifiers. Up-conversion and down-conversion were utilized for digitally reading the phase and amplitude of the signal. To obtain signals fromAttorney Docket No. 206085-0196-00WOfast moving cells, the time constant of the lock-in amplifiers was set as 501 ps, and the sampling rates were set as 14.39 kSa / s and 13.39 KSa / s for low-frequency sensing, and high-frequency sensing, respectively.

[0204] Signal obtained from the low-frequency part of the sensor provided the cell’s geometrical volume upon height calibration by the use of calibration particles (Polystyrene 20 pm, Sigma-Aldrich) added into the sample for this purpose. The phase and amplitude response of the microwave resonator were obtained from the high-frequency part of the sensor, and the out-of-phase component (Y = R sinO) of the reflected voltage, was calculated which probes any minute change in the capacitance of the resonator.Equation 1

[0205] Here, AY refers to the change in the out-of-phase component of the reflected voltage, C refers to the total capacitance of the resonator, and AC refers to the change of capacitance due to the passage of a cell. The change of phase (AO) and amplitude (AR) were extracted for transit of single cells through the sensing area. The capacitance change due to the presence of a cell can be calculated by:AY = ARsinO -I- Rcos(0)A0Equation 2where R and 0 refer to the baseline amplitude and phase values in raw signal.

[0206] The capacitance change calculated is a function of geometrical volume and the Clausius-Mosotti factor of the cell (a factor depending on the microwave permittivity values of the cell and the medium), decoupling the geometrical volume as obtained by the Coulter counter enables attaining the microwave permittivity of individual cells. The details of the circuitry, calibration procedure, and data analysis can be found in an earlier work (Tefek et al., 2023, Advanced Materials. 35:2304072). The Earth-Mover Distance calculations follow the standard definition (Kimmerling et al., 2022, Communications Biology. 5:1295; Orlova et al., 2016, PLoSAttorney Docket No. 206085-0196-00WOOne. 11 :e0151859) and the error bars are calculated using bootstrapping techniques using 200 resamples.

[0207] Cell culture and reagents: Human colorectal cancer cells (DLD1) were gifted by Prof. Alain Chariot, GIGA center, Belgium and cultured according to supplier’s protocol.HEK293 FT cells were cultured in Dulbecco’s Modified Eagles’ Medium (DMEM, Biowest), while DLD1 cells were cultured in RPMI 1630 media (Biowest). Subculturing and passaging were carried out using lx Trypsin EDTA (Biowest) and lx PBS (Biowest). Parental and cisplatin resistant HCC1937 cells were cultured in DMEM with 10% FBS, 50 U / mL penicillin / streptomycin, 1% nonessential amino acids (Gibco). Parental and tamoxifen resistant MCF-7 cells were cultured in phenol red-free DMEM (Gibco) with 10% FBS, 0.1% insulin, 50 U / mL penicillin / streptomycin, 1% nonessential amino acids (Gibco).

[0208] Hyper / Hypotonic PBS microwave measurements: Hypotonic solutions were prepared by taking commercial sterile lx PBS solution (Biowest) and diluting it using HPLC-grade water (Sigma-Aldrich) at 1 : 1, 1:4, and 1:10 PBS:water to gain solutions with 100% PBS concentration, 50%, 20%, and 10%, respectively. A 150% PBS solution was prepared by dissolving 16.9 mg / mL of pure KC1 in stock ( lx) PBS.

[0209] HEK293 FT were grown from frozen stock in 6-well plates in DMEM supplemented with 10% Fetal Bovine Serum (FBS). The cells were grown under incubation conditions for 48 h until reaching 80% confluency. After reaching confluency, the cells were trypsinized using a lx trypsin-EDTA solution for around 3 min, followed by gentle mixing and aspiration into 15 mL Falcon tubes. The cells were then centrifuged, the supernatant was removed, and 3 mL of fresh PBS solution of either 150, 100, 50, 20, or 10% (as outlined in the results section) was added. The cells were resuspended in the PBS solution through gentle mixing and aspiration. The cells were then injected into the microfluidic channel of the sensor for measurements.

[0210] Development of drug-resistant cell lines: Gefitinib was purchased from Santa Cruz Biotechnology, Inc and dissolved in cell culture-grade dimethyl sulfoxide (DMSO). DLD1 cells were continuously exposed to Gefitinib by gradually increasing the concentration over time. Since gefitinib was delivered in DMSO, control DLD1 cell population was cultured in parallel with equal amounts of DMSO without gefitinib, serving as the control cells. Gefitinib resistantAttorney Docket No. 206085-0196-00WODLD1 cell line was obtained when their LC50 to the drug reached double the concentration for that of the control cells. It took roughly one year starting from 12 gM, then the resistant cells were maintained in 30 gM Gefitinib. Cisplatin was obtained from NOVAGENTEK (Turkey) in powder form and working stock solutions were prepared in HPLC-grade water (Sigma-Aldrich). HCC-1937 cisplatin resistant cells were developed by culturing the cells in the presence of 7.5 gM, for over 6 months. The parental counterparts were cultured in the absence of cisplatin. Tamoxifen-resistant MCF-7 cells (MCF-7 TamR) were generated by culturing the cells in the presence of 5 gmol / L of 4-hydroxytamoxifen (Sigma-Aldrich) for over 1 year. In parallel, parental MCF-7 cells were maintained under identical conditions without tamoxifen.

[0211] Cell viability measurements: In parallel with sensor measurements, conventional cell viability measurements were conducted to validate the sensor data. In order to calculate the total cell death, we first calculated the total number of cells remaining in a given sample after the administration of the drug dose and incubation for 72 hours using manual cell counting on a haemocytometer in the presence of Trypan Blue. Cells that took up Trypan Blue were excluded from the total count since they were considered non-viable. The number of remaining cells in the given sample was then compared to the number of cells in the control sample (where 0 gM of the drug was administered). This yielded a percentage of the number of surviving cells in the measured sample relative to the control sample, which was then reported in the text.

[0212] Drug resistance tests- electronic measurement workflow: For microwave measurements of drug-resistant cells and their control ones, cells were seeded into 6-well plates (Greiner Bio One) to obtain 60% confluency next day and allowed to attach to the plates overnight under incubation conditions (37° C, 5% CO2,). The following day, the wells were replaced with fresh media with different drug concentrations as described in the figure legends. The plates were then incubated for 72 hours before initiating measurement procedures.

[0213] After 72 hours, each well was treated as follows: media containing the drug was aspirated and placed in a falcon tube. A trypsin solution was then added to the well and allowed to incubate at 37 °C for approximately 3 minutes. Upon cell detachment, the cells were resuspended in solution through gentle pipetting, and the cell suspension was then recombined with the previously aspirated media in the falcon tube. This was done to collect both floating and adherent cells in the well upon treatments. The collected cells were then centrifuged at 1500 refAttorney Docket No. 206085-0196-00WO(Eppendorf centrifuge) and the supernatant was discarded. Finally, each of the cell pellets were resuspended in 3 mL of 1 x PBS. 20 pL of the cell suspension was taken and mixed at a 1:1 ratio with filtered (0.22 pm polycarbonate filters) Trypan Blue dye for 1 min, followed by measuring cell viability as described previously using a haemocytometer. The remaining cell suspension was then mixed with 20 pm monodisperse polystyrene microsphere solution (Sigma-Aldrich, Product No: 74491) to serve as an internal calibration standard. The cell suspension was then introduced into the inlet of the microfluidic channel for the sensor device using a Fluigent pressure control system. The cells and polystyrene spheres were allowed to freely flow through the sensing region of the device and up to 500 individual measurement events were collected for each repetition. Three separate viability measurement repetitions were performed for each well. The outflow was then discarded appropriately.

[0214] Prior to the subsequent measurement, the microfluidic channel of the device was thoroughly washed with PBS and HPLC-grade water to prevent cross-contamination between samples.

[0215] Establishment and culture of PDOs: Patient-derived organoids (PDOs) were generated from a fresh surgical specimen obtained from a patient who underwent surgical resection at Hacettepe University Hospital. The study was approved by the Ethical Committee of Hacettepe University (reference number: KA-20136). PDO establishment and culture were performed as previously described (Vlachogiannis et al., 2018, Science. 259:920-926).

[0216] Briefly, fresh tissue was transferred into cold Dulbecco's Phosphate-Buffered Saline (DPBS; Sartorius) supplemented with IX Penicillin-Streptomycin (Thermo Fisher Scientific). The tissue was washed at least three times with cold DPBS, minced, and collected into 15 mL Falcon tubes. It was then conditioned with 5 mL PBS containing 5 mM EDTA (Sartorius) for 15 minutes at room temperature. Enzymatic digestion was performed using TrypLE™ (Thermo Fisher Scientific) for 1 hour at 37°C. To facilitate cell release, mechanical dissociation (pipetting) was applied. Dissociated cells were collected in Advanced DMEM / F12 (Thermo Fisher Scientific) and filtered through a 40 pm strainer (Greiner) to remove undigested tissue fragments.Attorney Docket No. 206085-0196-00WO

[0217] The filtered cells were centrifuged at 1200 rpm for 5 minutes at 4°C, resuspended in 120 pL growth factor-reduced (GFR) Matrigel (Corning), and seeded into a 24-well cell culture plate (Coming). The Matrigel was allowed to solidify for 20 minutes at 37°C in a 5% CO2 incubator, after which 500 pL of Advanced DMEM / F12 medium containing IX Penicillin-Streptomycin, 2 mM L-Glutamine (Thermo Fisher Scientific), 100 pg / mL Primocin (Invivogen), IX B27 supplement (Gibco), IX N2 supplement (Gibco), 0.01% BSA (Sigma), 50 ng / mL human EGF (Biolegend), 100 ng / mL human Noggin (Biolegend), 500 ng / mL human R-Spondin-1 (Biolegend), 10 nM Gastrin (Sigma Aldrich), 1 pM Prostaglandin E2 (Tocris Bioscience), 10 ng / mL human FGF-10 (Biolegend), 10 ng / mL human FGF-basic (Biolegend), 100 ng / mL human Wnt-3a (R&D Systems), 4 mM Nicotinamide (Sigma Aldrich), 0.5 pM A83-01 (Tocris Bioscience), and 5 pM SB202190 (Sigma Aldrich) was added. Additionally, the final medium was supplemented with 10 pM ROCK inhibitor Y-27632 (Sigma Aldrich).

[0218] PDO drug sensitivity measurement: For the drug assay, PDOs were mechanically harvested using 5 mL PBS / EDTA (1 mM) containing IX TrypLE™ (Gibco) and incubated for 10 minutes at 37°C. PDOs were then mechanically dissociated into single cells, washed with Hank's Balanced Salt Solution (HBSS; Thermo Fisher Scientific), pelleted at 1200 rpm for 5 minutes at 4°C, resuspended in 120 pL GFR Matrigel, and reseeded at a density of 1 x 105cells per well into a 24-well plate. Following Matrigel solidification for 20 minutes at 37°C in a 5% CO2 incubator, 500 pL of complete organoid medium was added to each well. The medium was refreshed after 24 hours.

[0219] Four days post-seeding, 1 pM Gefitinib (Selleckchem), 1 pM Bortezomib (Selleckchem), or DMSO (control) was added to the wells. After 6 days of drug treatment, PDOs were dissociated into single cells using IX TrypLE™, pelleted at 1200 rpm for 5 minutes at 4°C, resuspended in HBSS, and introduced into the microfluidic device channels for further analysis.

Claims

Attorney Docket No. 206085-0196-00WOCLAIMSWhat is claimed is:

1. A system for measuring the geometric diameter and electrical diameter of a cell, comprising:a planar body with a microchannel comprising an inlet and an outlet, wherein the microchannel comprises a centrally located constriction;a split-ring resonator (SRR) positioned on the body comprising an inner ring and an outer ring, wherein the inner ring comprises a gap in the ring; anda Coulter counter positioned on the body comprising first and second electrodes, wherein the constriction of the microchannel, the gap of the inner ring, and the first and second electrodes of the Coulter counter are aligned to form a sensing region for measuring a sample comprising at least one cell.

2. The system of claim 1, wherein the inner ring comprises a first end comprising a tabbed portion, and a second end comprising a slotted portion, wherein the tabbed portion at least partially extends into the slotted portion to form the gap.

3. The system of claim 2, wherein the tabbed portion is asymmetrically positioned in the slotted portion.

4. The system of claim 3, wherein the width of the inner ring tapers from the second end to the first end.

5. The system of claim 1, wherein the width of the inner ring is less than the width of the outer ring.

6. The system of claim 1, wherein the first and second electrodes of the Coulter counter are separated by a distance along the length of the body ranging between 10 pm and 1000 pm.Attorney Docket No. 206085-0196-00WO7. The system of claim 1, further comprising a signal generator and a first lock-in amplifier electrically connected to the outer ring of the SRR, and a second lock-in amplifier electrically connected to the electrodes of the Coulter counter.

8. The system of claim 1, wherein the SRR is a high-frequency sensor and the Coulter counter is a low-frequency sensor.

9. The system of claim 1, wherein the sample comprising at least one cell is passed from the inlet to the outlet across the sensing region to measure the geometric diameter and electrical diameter of the at least one cell.

10. A method of measuring the geometric diameter and electrical diameter of a sample comprising at least one cell using the system of claim 1.

11. A method of identifying a population of cells as susceptible or resistant to a drug comprising measuring the geometric diameter and electrical diameter of the at least one cell.

12. The method of claim 11, wherein the method comprises exposing the at least one cell to a given dose of the drug and subsequently measuring the geometric diameter and electrical diameter of the at least one cell.

13. The method of claim 12, wherein the method comprises:a) providing a system comprising:a planar body with a microchannel comprising an inlet and an outlet, wherein the microchannel comprises a centrally located constriction;a split-ring resonator (SRR) positioned on the body comprising an inner ring and an outer ring, wherein the inner ring comprises a gap in the ring; anda Coulter counter positioned on the body comprising first and second electrodes, wherein the constriction of the microchannel, the gap of the inner ring, and the first and second electrodes of the Coulter counter are aligned to form a sensing region; andAttorney Docket No. 206085-0196-00WOb) contacting the at least one cell to the sensing region.

14. The method of claim 13, wherein the method comprises applying a high-frequency signal to the SRR and obtaining the change in capacitance at the gap in the inner ring to measure the electrical diameter of the at least one cell, and applying a low-frequency signal to the Coulter counter and obtaining the change in current across the electrodes to measure the geometric diameter of the at least one cell.

15. The method of claim 14, wherein the method comprises comparing the geometric diameter and electrical diameter of the at least one cell exposed to the drug with a baseline measurement.

16. The method of claim 15, wherein the baseline measurement is the geometric diameter and electrical diameter of the at least one cell without being exposed to the drug.

17. The method of claim 15, wherein the cell is determined to be susceptible to the given dose of the drug when the geometric diameter is increased or decreased by at least 5%.

18. The method of claim 15, wherein the cell is determined to be susceptible to the given dose of the drug when the electrical diameter is increased or decreased by at least 5%.

19. The method of claim 11, wherein the cell is a cancer cell.

20. The method of claim 19, wherein the cell is a tumor cell.

21. The method of claim 11, wherein the drug is a cytostatic, cytotoxic, or mixed-effect drug.

22. A method of treating cancer in a subject comprising determining that a population of cancer cells from the subject is susceptible to a drug by the method of claim 10, and administering the drug to the subject.

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