Whole cell assays and methods

Abstract

The disclosure provides methods for analysis of disease cell response to a therapeutic agent. In embodiments, a method comprises administering the therapeutic agent to a disease cell sample from the subject in a device that measures at least one physiological parameter of a cell; determining whether a change occurs in the physiologic parameter of the disease cell sample in response to the therapeutic agent as compared to a baseline measurement or the physiological parameter before administration of the therapeutic agent, and selecting the therapeutic agent that results in the change in the at least one physiologic parameter. In embodiments, the disease cells are whole, viable, and/or label free.

1 . A method for selecting a treatment for an individual subject comprising:
 a) administering a targeted therapeutic agent that has demonstrated efficacy for cancer to at least one isolated label free cancer sample consisting essentially of viable diseased cells extracted from the individual subject's specimen in a cellular response measurement system (CReMS), wherein the efficacy of the targeted therapeutic agent for the individual subject is unknown;   b) continuously monitoring the intended agonistic or antagonistic effect of the targeted therapeutic by measuring at least one physiological response parameter of the cell sample while the cells are still viable for a defined period of time sufficient to detect any non-linear Euclidean distance change, wherein the physiological response parameter is selected from the group consisting of cell adhesion, cell attachment, cell morphology, cell size, cell shape, cell phenotype, cell proliferation, cell signaling, cell density, cell polarity, pH, O2, CO2, glucose, distinctive protuberances, outgrowths, spreading, foci density, cytoskeletal arrangements, cell proliferation patterns, receptor phagocytosis, attachment foci number, uptake or efflux of metabolites, signaling proteins, growth factors, ATP, amine containing materials such as proteins, amino acids, extracellular matrix, or cell signaling molecules, ions such as magnesium, calcium, potassium, and combinations thereof; and   c) analyzing the change in the physiological response parameter of the viable cells in the cell sample by performing a nonlinear Euclidean distance change analysis to generate a value for that cell sample when in the presence of the targeted therapeutic agent and comparing the value to a baseline value; wherein the change in the value for that cell sample as compared to the baseline value is predictive of the efficacy of the targeted therapeutic agent for the disease in the individual subject.   
     
     
         2 . The method of  claim 1 , wherein the therapeutic agent is one or more agents. 
     
     
         3 - 4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the agent is targeted to a cellular response pathway, or targeted to a specific cellular molecule. 
     
     
         6 . The method of  claim 1 , wherein the targeted therapeutic agent is selected from the group consisting of trastuzumab, pertuzumab, lapatinib, docetaxel, tamoxifen, abraxane, paclitaxel injection, brentuximab vedoton, everolimus, pemetrexed, exemestane, ofatumumab, bevacizumab, alemtuzumab, irinotecan, bicalutamide, cetuximab, visomedegib, toremifene citrate, fulvestrant, imatinib, topeotecan, axitinib, romidepsin, cabrazitaxel, sorafenib, infliximab, lenalidomide, rituximab, dasatinib, sunitinib, erlotinib, nilotinib, paclitaxel, panitumumab, bortezomib, pazopanib, crizotinib, ipilimumab, vemurafenib, goserelin acetate, a BH3 mimetic, navitoclax, anastrozole, letrozole, an aromatase inhibitor, ixabepilone, aflibercept, temsirolimus, irbritumomab, abiraterone, custirsen, neratinib, and combinations thereof. 
     
     
         7 . The method of  claim 1 , further comprising administering at least one activator agent that perturbs a cellular response pathway to the isolated label free sample in a cellular response measurement system prior to or after administering the therapeutic agent. 
     
     
         8 . The method of  claim 7 , wherein the activator agent is targeted to a cellular response pathway comprising MAPK, RHO, AKT, FAK1, RAS/RAF, PI3K/PTEN, MAK, MKK, MEK, mTOR, NOTCH, WNT, Jak/STAT, HIF1α, Hedgehog, TGFβ, growth factor, cell adhesion, signaling, apoptotic pathways or combinations thereof. 
     
     
         9 . The method of  claim 8 , wherein the activator agent is targeted to a cell surface receptor or targeted to a specific cellular molecule comprising EGFR, EGFR-TK, PI3K, MEK1, MEK2, HER2 receptor, Her3 receptor, Her4 receptor, VEGFR, Topoisomerase I, TUBB1, BCL2, TGFR, TNFR, FGFR, MAPK, MAK, serine threonine protein kinases, tyrosine kinases, FAS death receptor, NOTCH, WNT, Jak/STAT, HIF1α, Hedgehog, Nerve Growth Factor Receptor, Hepatocyte Growth Factor Receptor, Interleukin Receptor, DNA, purine crosslinking agents, thymidylate synthase, cell adhesion, signaling and apoptotic pathways or combinations thereof. 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 1 , wherein the cancer cells are breast, colon or lung cancer cells. 
     
     
         12 . The method of  claim 1 , wherein the cellular response measurement system (CReMS) comprises a biosensor. 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 12 , wherein the device is an impedance or an optical device. 
     
     
         15 . The method of  claim 1 , wherein the baseline is made from healthy cells from the same subject. 
     
     
         16 . The method of  claim 1 , wherein the baseline is made from diseased cells from the same subject or from diseased of other patients known either to respond or not to respond to the agent. 
     
     
         17 . A method for selecting a treatment for an individual subject having cancer of  claim 1  further comprising
 d) selecting the targeted therapeutic agent that is predicted to have efficacy for the disease in the individual subject for treatment of the subject. 
 
     
     
         18 - 24 . (canceled) 
     
     
         25 . The method of  claim 17 , wherein the cancer cells are breast, colon or lung cancer cells. 
     
     
         26 - 30 . (canceled) 
     
     
         31 . A method for selecting a treatment for an individual subject comprising:
 a) administering a targeted therapeutic agent that affects a cellular pathway to at least one isolated label free cancer sample consisting essentially of viable diseased cells extracted from the individual subject's specimen in a cellular response measurement system (CReMS), wherein the efficacy of the targeted therapeutic agent for the individual subject is unknown;   b) administering an activating agent known to perturb the cellular pathway of the cell sample to at least one isolated label free cancer sample in a cellular response measurement system prior to, at the same time as, or after administering the targeted therapeutic agent;   c) continuously monitoring an intended agonistic or antagonistic effect of the targeted therapeutic by measuring at least one physiological response parameter of the cell sample for a defined period of time sufficient to detect a nonlinear change while the cells are still viable in the presence of the combination of the targeted therapeutic and activator agents, wherein the physiological response parameter is selected from the group consisting of cell adhesion, cell attachment, cell morphology, cell size, cell shape, cell phenotype, cell proliferation, cell signaling, cell density, cell polarity, pH, O2, CO2, glucose, distinctive protuberances, outgrowths, spreading, foci density, cytoskeletal arrangements, cell proliferation patterns, receptor phagocytosis, attachment foci number, uptake or efflux of metabolites, signaling proteins, growth factors, ATP, amine containing materials such as proteins, amino acids, extracellular matrix, or cell signaling molecules, ions such as magnesium, calcium, potassium, and combinations thereof; and   d) analyzing the nonlinear change in the physiological response parameter of the viable cells in the cell sample by performing a nonlinear analysis to generate a value for that cell sample when in the presence of a combination of the activator agent and targeted therapeutic agent and comparing the value to a value of a cell sample in the presence of the activator agent or targeted therapeutic agent alone; wherein the change in the value for that cell sample as compared to the value of a cell sample in the presence of the activator agent or targeted therapeutic agent alone is predictive of the efficacy of the therapeutic agent for the disease in the individual subject.   
     
     
         32 . The method of  claim 31 , wherein the targeted therapeutic agent is targeted to a cellular response pathway, or targeted to a specific cellular molecule. 
     
     
         33 . The method of  claim 31 , wherein the targeted therapeutic agent is selected from the group consisting of trastuzumab, pertuzumab, lapatinib, docetaxel tamoxifen, abraxane, paclitaxel injection, brentuximab vedoton, everolimus, pemetrexed, exemestane, ofatumumab, bevacizumab, alemtuzumab, irinotecan bicalutamide, cetuximab, visomedegib, toremifene citrate, fulvestrant, imatinib, topeotecan axitinib, romidepsin, cabrazitaxel sorafenib, infliximab, lenalidomide, rituximab, dasatinib, sunitinib, erlotinib, nilotinib, paclitaxel, panitumumab, bortezomib, pazopanib, crizotinib, ipilimumab, vemurafenib, goserelin acetate, a BH3 mimetic, navitoclax, anastrozole, letrozole, an aromatase inhibitor, ixabepilone aflibercept, temsirolimus, irbritumomab, abiraterone, custirsen, neratinib, and combinations thereof. 
     
     
         34 . The method of  claim 31 , wherein the activator agent is targeted to a cellular response pathway comprising MAPK, RHO, AKT, FAK1, RAS/RAF, PI3K/PTEN, MAK, MKK, MEK, mTOR, NOTCH, WNT, Jak/STAT, HIF1α, Hedgehog, TGFβ, growth factor, cell adhesion, signaling, apoptotic pathways or combinations thereof. 
     
     
         35 . The method of  claim 31 , wherein the activator agent is targeted to a cell surface receptor or targeted to a specific cellular molecule comprising EGFR, EGFR-TK, PI3K, MEK1, MEK2, HER2 receptor, Her3 receptor, Her4 receptor, VEGFR, Topoisomerase I, TUBB1, BCL2, TGFR, TNFR, FGFR, MAK, serine threonine protein kinases, tyrosine kinases, FAS death receptor, NOTCH, WNT, Jak/STAT, HIF1α, Hedgehog, Nerve Growth Factor Receptor, Hepatocyte Growth Factor Receptor, Interleukin Receptor, DNA, purine crosslinking agents, thymidylate synthase, cell adhesion, signaling, and apoptotic pathways or combinations thereof. 
     
     
         36 . The method of  claim 31 , wherein the cancer cells are breast, colon, or lung cancer cells. 
     
     
         37 . The method of  claim 31 , wherein the cellular response measurement system (CReMS) comprises a biosensor. 
     
     
         38 . (canceled) 
     
     
         39 . The method of  claim 31 , wherein the baseline is made from healthy cells from the same subject. 
     
     
         40 . The method of  claim 31 , wherein the baseline is made from diseased cells from the same subject or from diseased cells of other patients known either to respond or not to respond to the agent. 
     
     
         41 . The method for selecting a treatment for an individual subject having cancer of  claim 31  further comprising: e) selecting the therapeutic agent that is predicted to have efficacy for the disease in the individual subject for treatment of the subject. 
     
     
         42 . The method of  claim 1 , wherein the distance change is selected from the group consisting of nonlinear Euclidean comparisons of the test well against a positive or negative control, arithmetic summation of the difference at multiple time points, temporal maxima, temporal minima, time to reach maxima or minima, changes in slope, absolute drop in biosensor signal, a total of all measurements, and combinations thereof. 
     
     
         43 . The method of  claim 31 , wherein the distance change is selected from the group consisting of nonlinear Euclidean comparisons of the test well against a positive or negative control, arithmetic summation of the difference at multiple time points, temporal maxima, temporal minima, integrated areas under a curve, time to reach maxima or minima, derivatives of the temporal time course, changes in slope, second derivative of the time versus biosensor signal, absolute drop in biosensor signal, time for slope to change from positive to negative or negative to positive, a total of all measurements, and combinations thereof. 
     
     
         44 . The method of  claim 1 , further comprising deriving a final value which relates to a cutoff value above which and below which the efficacy of the therapeutic agent is predicted to be different by comparing values derived from nonlinear Euclidean comparisons of a test well against a positive or negative control, arithmetic summation of the difference at multiple time points, temporal maxima or minima time to reach maxima or minima, changes in slope, a total of all measurements, or combinations thereof, to a baseline value. 
     
     
         45 . The method of  claim 31 , further comprising deriving a final value which relates to a cutoff value above which and below which the efficacy of the therapeutic agent is predicted to be different by comparing values derived from nonlinear Euclidean comparisons of a test well against a positive or negative control, arithmetic summation of the difference at multiple time points, a temporal maxima or minima, integrated area under a curve, time to reach maxima or minima, derivatives of the temporal time course, changes in slope, second derivative of the time versus biosensor signal, time for slope to change from positive to negative or negative to positive, a total of all measurements, or combinations thereof, to a baseline value. 
     
     
         46 . The method of  claim 1 , wherein the cancer cells are prostate cancer cells. 
     
     
         47 . The method of  claim 31 , wherein the cancer cells are prostate cancer cells.