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WO1990011371A1 - PROCEDE ET SYSTEME DE DETERMINATION A L'ETAT NATIF DE LA VIABILITE ET DE LA CAPACITE DE PROLIFERATION DE TISSUS $i(IN VITRO) - Google Patents

PROCEDE ET SYSTEME DE DETERMINATION A L'ETAT NATIF DE LA VIABILITE ET DE LA CAPACITE DE PROLIFERATION DE TISSUS $i(IN VITRO) Download PDF

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Publication number
WO1990011371A1
WO1990011371A1 PCT/US1989/003719 US8903719W WO9011371A1 WO 1990011371 A1 WO1990011371 A1 WO 1990011371A1 US 8903719 W US8903719 W US 8903719W WO 9011371 A1 WO9011371 A1 WO 9011371A1
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WIPO (PCT)
Prior art keywords
tumor
cells
proliferation
marker
dna
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PCT/US1989/003719
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English (en)
Inventor
Kenneth M. Connors
Ann Z. Meerson-Monosov
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Anticancer Inc.
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Publication date
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Publication of WO1990011371A1 publication Critical patent/WO1990011371A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to a method of using an in vitro culture system to measure the cell proliferation and cell viability of human tissues, particularly tumor tissues, and to measure the efficacy of anti-neoplastic drugs upon the proliferation and viability of the cultured cells.
  • Background Cancer is a disease involving inappropriate cell division.
  • a realistic model is greatly needed to understand the biology of altered proliferation in cancer as compared to normal tissue and to use information on proliferation capacity as a basis of cancer prognosis and treatment.
  • TKI thymidine-labeling index
  • the nuclear antigen Ki-67 seems to be present in proliferating breast cancer cells [McGurrin et al., Cancer. 59:1744-50 (1987)], but its relevance to other tissue types is not yet known.
  • the present invention provides in improved in vitro assay system to determine cellular proliferation and/or cellular viability of cells from patient biopsies. It has been found that the determinations as to cellular proliferation and viability made according to the present invention accurately predict the grade, stage and overall aggressiveness of a tumor.
  • a method for measuring the tumor specific effects of an agent on cell proliferation and/or viability comprises histoculturing, in separate containers, first and second portions each of tumor tissue and normal tissue samples.
  • the normal tissue sample is from a tissue analogous to the stem cells of the tumor.
  • the histocultured samples are exposed to one or more concentration of an agent whose effects are being examined.
  • the exposed samples are then treated (contacted) with either a proliferation marker (DNA synthesis marker) and cultured (histocultured) for a predetermined period of time.
  • the percent of cell having marker incorporated therein is then determined, and, by comparing the results obtained from the tumor cells with the analogous normal cells, the tumor specific effect of the agent on cell viability and/or proliferation is determined.
  • the present invention provides a method for measuring the effect of an agent on the viability and/or proliferation of cells in their native environment, i.e., as part of a tissue.
  • the effect on the cells of the tissue to exposure to one or more given dose levels of one or more given drugs is measured by individually culturing aliquots of the same explanted tissue using a plurality of containers.
  • One of the aliquots serves as a control and is quantitatively assayed for cell viability and/or proliferation in the absence of drug exposure.
  • Each of the other aliquots is exposed to the drug at a different dose level and then quantitatively assayed for viability and/or proliferation using a protein-synthesis marker or DNA-synthesis marker, respectively.
  • the number of viable cells in each tissue sample is indicated by the number of cells having metabolically incorporated the protein-synthesis marker into the cellular protein.
  • the number of proliferating cells is indicated by the number of cells having metabolically incorporated the DNA-synthesis marker- into the cellular DNA.
  • Metabolic markers for cellular protein synthesis are well known in the art and include radioactively labeled amino acids such as 35 S- methionine, 14 C-alanine, 14 C-glycine, 14 C-glutamic acid, 14 C-proline, 3 H-leucine, 3 H-serine and the like.
  • Metabolic marker for cellular DNA- synthesis are also well known in the art and include radioactively labeled nucleotides such as H-deoxythymidine, 3 H-deoxyadenine, 3 H-deoxyguanine, 3 H-deoxycytosine and the like.
  • the various types of tissues to which the present invention is applicable include normal tissues as well as primary or metastatic tumors, including solid tumors (both carcinomas and sarcomas) and the like.
  • carcinomas include, for example, adrenal, bladder, breast, colon, kidney, lung, ovary, pancreas, prostate, thyroid, upper airways (head and neck) , uterus (corpus and cervix) , bile ducts, choriocarcino a, esophagus, liver, parathyroid, rectum, salivary glands, small bowel, stomach, testis, tongue and urethra.
  • sarcomas and other neoplasms include, for example, diffuse lymphomas, Ewing's tumor, Hodgkin's disease, melanoma (melanotic and amelanotic) , multiple myeloma, nephroblastoma (Wil 's tumor), neuroblastomas, nodular lymphomas, rhabdomyosarcoma, angiosarcoma, brain tumors (gliomas) , chondrosarcoma, dysgerminoma, fibrosarcoma, leiomyosarcoma, liposarcoma, medulloblastoma, mesothelioma, osteosarcoma, retinoblastoma and thymoma.
  • the tissue whose cell viability and/or proliferation characteristics are to be determined is explanted by an a septic surgical procedure and a portion thereof is divided into sections having a volume of about 0.5 to about 10, preferably a volume of about 1.0 to about 8.0, more preferably 1.0 to 2.0 cubic millimeters.
  • the explanted tissue is divided into aliquots, typically at least about six, one of which is typically designated a control that receives no exposure or contact with the compound being examined.
  • the aliquots are then histocultured on a hydrated extracellular-matrix- containing gel so that the three-dimensional integrity of the tissue is maintained.
  • Tissue culture grade extracellular- matrix- containing gels suitable for histoculture are well known in the art and include those described by U.S. Patent No. 4,060,081 to Yannas et al., that available under the tradena e of Gelfoam from Upjohn, and the like.
  • liquid tissue culture nutrient media capable of supporting tissue cell growth are known in the art.
  • the medium used can either be serum-containing or serum-free with additives such as insulin, transferrin, selenium, estradiol and the like.
  • a culture medium found to be particularly suitable in the present invention in Eagle's minimal essential medium (MEM) [Eagle, Science. 122:501 (1955) and Eagle, Science, 130:432 (1959) ].
  • the tissues are typically histocultured for at least 2-5 days preferably about 3-4 days prior to exposing the cells therein to the agent being examined. Culturing is typically performed in a humidified atmosphere at a temperature corresponding to that of the body temperature of the animal from which the tissue sample came, e.g. 37'C for human tissue samples.
  • Drug exposure of the cells for the purposes of the viability and/or proliferation measurements is preferably carried out prior to treating the tissue samples with the protein- synthesis and/or DNA-synthesis markers.
  • the procedure involves incubating the tissue sample with a predetermined amount (determinate concentration) of the drug for a predetermined period of time (determinate time period) and thereafter separating the tissue sample from the drug, and preferably washing the tissue sample free of residual drug.
  • drug exposure dose level refers to the quantitative product of the drug concentration (e.g. in ⁇ l) and the time of the exposure period (e.g., in hours or minutes).
  • the drug concentrations and exposure times are typically calculated from pharmacological data to the simulate in vitro the drug exposure dose level achieved in vivo.
  • the drug exposure dose level required in carrying out the drug sensitivity measurements in accordance with the assay of the present invention is at a maximum of only 5 to 10% of the clinically achievable drug exposure dose level for the known anticancer drugs which have been tested in the present system.
  • the drug sensitivity measurements as described above can be carried out in a manner which enables the determination, for any given drug, of a "drug sensitivity index", which is indicative of the antineoplastic activity of the given drug against the specific human tumor from which the explanted cells were obtained.
  • This procedure involves carrying out the drug sensitivity measurements of a plurality of dose levels extending over a multi-log range, and then using the results of these measurements to plot a curve of percent survival (the percentage of the assay count resulting from drug exposure versus the assay count of the control in the absence of drug exposure) versus drug exposure dose level.
  • the "drug sensitivity index" of the given drug is then quantitated by measuring the area under such curve out to a defined upper limit which is correlated to the clinically achievable peak drug exposure dose level for that drug.
  • the sensitivity index obtained in the above-described manner is highly indicative of the antineoplastic activity of the drug against the specific human tumor from which the explanted cells were obtained, with a low sensitivity index indicating high antineoplastic activity.
  • the samples are treated (cultured in the presence of) the proliferation and/or viability marker.
  • the samples After the cells of the tissue samples have been exposed to the drug(s) , treated with the proliferation and viability markers and subsequently histocultured, the samples are typically fixed in the tissue fixative such as formalin, embedding in paraffin or the like and sectioned on a microtine.
  • the viability and/or proliferation markers contain radioactive labels, the sections are then treated with nuclear- track emulsion such as NTB (Kodak) , and developed.
  • the sections are then assayed for the percentage of cells positive for the presence of the viability of proliferation marker.
  • radioactive labels and nuclear-track emulsions are used, this can be accomplished by light-polarized microscopy.
  • the magnified image can be digitized by processing it through a video camera operatively linked to a computer capable of digitizing the image for analysis.
  • the in vitro assay system of the present invention has utility for the in vitro prediction of clinical response to cancer chemotherapy, as well as the screening of new anticancer drugs for clinical trial.
  • the explanted cells obtained from a biopsy of such specific tumor can be assayed in accordance with the present technique, a drug sensitivity measurements can be carried out for a plurality of different anticancer drugs which are potentially clinically effective for the chemotherapeutic treatment of the specific tumor.
  • these sensitivity indices may be used for predictably selecting the most promising effective of the drugs to be used for the chemotherapeutic treatment.
  • the present invention contemplates a method of determining the "growth factor index" of a tumor.
  • the growth factor index of a tumor correlates to the tumor's in vivo grade and stage, i.e., the in vivo aggressiveness of the tumor, particularly in breast and ovarian carcinomas.
  • the growth factor index is determined by histoculturing, as previously described, a sample of tumor cells.
  • the cells are then treated with a proliferation marker such as one of the before- described DNA-synthesis marker, e.g., 3 H- deoxythymidine.
  • the treated cells are cultured for a predetermined period of time and then the percent of sample cells containing the proliferation marker incorporated into the cellular DNA is determined, that percentage representing the tumor's intrinsic growth factor index.
  • the present invention contemplates a kit for the in vitro determination of viability and/or proliferation of cells as described herein.
  • the kit contains, in an amount sufficient for at least one assay, an agent to be tested for its effects on viability and/or proliferation, a viability and/or proliferation marker, and a container in which to perform the assay.
  • MEM Eagle's minimal essential medium
  • glutamine 10% fetal calf serum
  • nonessential amino acids nonessential amino acids
  • the antibiotics garamycin and claforan was added to the cultures such that the upper part of the gel was not covered, and cultures were maintained at 37 degrees C in a carbon dioxide incubator to allow the explanted tissue specimens to grow.
  • Cellular DNA is labeled in any cells undergoing replication within the tissues. After 4 days of labeling, the cultures were washed with phosphate-buffered saline, placed in histology capsules, and fixed in 10% Formalin. The cultures were then dehydrated, embedded in paraffin, and sectioned by standard methods, and the sections were placed on slides. The sections on the slides were then deparaffinized and prepared for autoradiography by coating with Kodak NTB-2 emulsion in the dark and exposed for 5 days, after which they were developed. After developing and rinsing, the sections were stained with hematoxylin and eosin.
  • the sections were then analyzed by determining the percentage of cells undergoing DNA synthesis in the various tissue cultures, using a Nikon or Olympus photomicroscope fitted with an epi-illumination polarization lighting system. Replicating cells were identified by the presence of silver grains, visualized as bright green in the epi-illumination polarization system, over their nuclei due to exposure of the NTB-2 emulsion to radioactive DNA. The above procedures produce a histological autoradiogram showing cellular proliferation of the cells in a tumor explant tissue.
  • Tumors tested include tumors of the colon, ovaries, pancreas, bladder, kidney, brain, and parotid, and also include small- cell lung carcinoma and Ewing sarcoma. In all cases, three-dimensional tissue organization representative of the original tissue is maintained throughout the culture period. A high degree of detection of radiolabeled proliferating cells is afforded by the epi-illuminescence polarization microscopy, which enhances detection of the autoradiographic exposed silver grains by the scatter of incident polarized light.
  • the proliferation capacity of a metastatic colorectal tumor exhibited high labeling in culture. More than 90% of the cells in the observed culture preparation had proliferated during the labeling period of this relatively undifferentiated colon metastasis to the liver.
  • the proliferation capacity of a small- cell lung tumor exhibited the maintenance of the two major classes of oat cell types: the classic small cells and the more elongate fusiform cell types, each having a high degree of cell proliferation.
  • the proliferation capacity in ovarian carcinoma consistently exhibited an extremely high index of proliferation of the epithelial cells while the stromal cells remained quiescent.
  • the histological autoradiogram showed the high proliferative capacity of the ovarian carcinoma cells which have invaded the supporting gel matrix.
  • This invasive behavior may mimic the way ovarian tumors frequently invade the peritoneal wall in vivo.
  • the proliferation capacity in miscellaneous tumors including those of the pancreas, bladder, kidney, brain, and parotid gland, and a Ewing sarcoma exhibited the intricate gland formations containing proliferating cells -in many of these cultured tumors. It is important to note that distinctions can be observed in the prepared autoradiograms between proliferating malignant cells and normal cell types, such as for the breast tumor epithelial cells and normal stromal cells. An additional important observation in these studies is that normal tissues culture and proliferate well. Explanted tumor and adjacent normal tissue from the breast of patient 431 were compared.
  • This native-state culture system allows a high probability of detecting potential proliferative cells.
  • a video camera was attached to the microscope. Autoradiograms prepared as above using breast carcinoma tissue were then viewed under polarizing light without bright-field light, thereby visualizing only the radioactive cells which have exposed silver grains of the nuclear- track emulsion. The radioactive cells brightly reflect the polarized light.
  • the resulting image was analyzed by a computer-assisted image analysis apparatus in which the image was first digitized by a digitizer board, and then the area of brightness corresponding to the number of labeled or bright cells was calculated as the area of enhanced pixels by the Fas-Corn version of the P-See program (The Microworks, Del Mar, CA) run on an IBM PC XT compatible computer.
  • the area of enhanced pixels is proportional to the number of labeled cells.
  • the image analysis system the autoradiograms were automatically analyzed for the number of labeled proliferating cells.
  • the labeled cells of a cultured breast tumor appear bright green.
  • epi-illumination polarization microscopy using polarized light without bright- field only the labeled cells were visualized.
  • the image of the labeled cells was then digitized through a video camera and the P-See program.
  • the area of brightness or enhanced pixels was then automatically determined by the Fas-Corn program.
  • the area of enhanced pixels is proportional to the number of labeled cells, enabling the automatic counting of labeled, proliferating cells.
  • Tumor tissue specimens from a patient having breast carcinoma were obtained as described in Example 1, divided into 1 mm diameter pieces, and were each placed onto a flexible gel matrix to form a three dimensional culture. Duplicate cultures are prepared of each specimen, and 8 microcuries (uCi) of either 35S-methionine or 3 H- thymidine was added to each culture that includes 2 milliliters (ml) of culture medium containing the added radiolabel.
  • the labeled cultures were maintained as before for four days, the excess radiolabel was then rinsed off of each cultured specimen using a series of phosphate-buffered saline (PBS) rinses and the cultures were each processed for histological and autoradiographic visualization as described in Example 1.
  • PBS phosphate-buffered saline
  • the prepared cultures were then analyzed by using the computer program Fas-Corn for analysis after digitizing as described in Example 1.
  • the measure of 35S-methionine incorporated into cultured cells allows the determination of cellular protein synthesis and is therefore a measure of cell viability.
  • the measure of 3 H-thymidine incorporation into cultured cells allows the determination of DNA synthesis and is therefore a measure of cell proliferation.
  • Tissue specimens cultured in the presence of 35S-methionine or H-thymidine incorporated radiolabel in the portions of the tissue containing viable or proliferating cells, respectively, or both.
  • Non-viable or non-proliferating cells did not incorporate their respective labels and did not present silver grains on visual inspection of prepared specimens, nor present proliferating cells as bright green objects when analyzed in the epi- illumination polarization system.
  • the extent of proliferation measured by the in vitro native-state culturing system correlates with the grade and stage of the tumor: the more malignant the tumor, the higher the proliferation measured in vitro.
  • the extent of proliferation can be expressed as a growth fraction index (GFI) , measured as the percentage of proliferating cells present in a population of the total number of cells present in a selected field viewed by the microscope. Therefore measured GFI can be used to predict the clinical progression of the human cancer tissue tested.
  • GFI growth fraction index
  • the Fas-Corn program analysis provides a quantitative means that is semi-automated to determine the proliferative capacity or viability of a tumor, and is ideally suited to provide GFI data. Proliferation analyses were conducted on numerous explanted breast tumor tissues graded as metastatic or primary, and also graded as poorly or moderately differentiated to generate an average GFI for each type of tumor tested.
  • metastatic tumors averaged 0.437 + 0.149 GFI
  • primary tumors exhibited a lower average of 0.282 + 0.138 GFI.
  • poorly differentiated tumors averaged 0.372 + 0.150 GFI
  • moderately differentiated tumors averaged 0.220 + 0.094 GFI. The results indicate that GFI correlates with tumor severity and clinical prognosis.
  • Tissue specimen cultures were established using various tumor tissues as described in Example 2. After the fourth day of culturing, multiple cultures of each tissue were further cultured in the presence of a drug indicated below at the indicated concentrations for an exposure time of 24 hours.
  • Cultures were then washed in culture medium to remove excess drug, cultured in the absence of drug for 3 days to allow the cells to recover from transient drug effects, and were then labeled as described in Example 2. After labeling, the cultures were processed as before in Example 1 to visualize the degree of viability and proliferative capacity in the specimens when cultured in the presence of the drug.
  • the cells cultured from the ovarian carcinoma tissue of a patient having ovarian carcinoma were tested by the above methods for cell proliferation in the presence of cisplatin at 1.5 ⁇ g/ml, 5-fluorouracil at 4 ⁇ g/ l, melphalan at 10 ⁇ g/ml, methotrexate at 22.5 ⁇ g/ml or thiotepa at 60 ⁇ g/ml.
  • the results showed a decrease in the detectable signal generated by both H-thymidine and 35S-methionine incorporation, indicating an inhibition of both proliferation and viability.
  • a diminution in cell proliferation is expressed as a decrease in the GFI, when compared in the GFI for the same specimen cultured in the absence of the drug.
  • the cells cultured from the tissue of a patient having breast carcinoma were tested as above in the presence of drug, and resulted in the following diminutions in cell proliferation shown in the parenthesis: Adriamycin at 290 ng/ l (90%) , 5-fluorouracil at 4 ⁇ g/ml (90%) , melphalan at 1 ⁇ g/ml (80%), methotrexate at 2.25 ⁇ g/ml (70%) or. vincristine at 23 ⁇ g/ml (70%) .
  • Patient D.H. was diagnosed as having breast carcinoma and was determined to be non- responsive to in vivo therapy with either 5- fluorouracil or adriamycin. Cellular proliferation of patient D.H.
  • the cells cultured from the cancerous tissue from a patient having colon/rectal cancer were tested as above in the presence of the various indicated drugs, and resulted in the following diminutions in cell proliferation shown in the parenthesis: 5-fluorouracil at 4 ⁇ g/ml (90%) , mitomycin C at 1 ⁇ g/ml (90%) , and BCNU at 2 ⁇ g/ml (90%).
  • Patient V.S. was diagnosed as having colon carcinoma and was determined to be non-responsive to in vivo therapy using 5-fluorouracil.
  • Cellular proliferation of V.S.'s colon carcinoma cells was not inhibited significantly, i.e., greater than 90%, by culturing the explants as above in the presence of 5-fluorouracil.
  • a clinical correlation was again observed between in vivo and in vitro responsiveness.
  • cellular proliferation can be used as a measure of a tumor's drug responsiveness, where an active drug inhibits cellular proliferation.
  • the viability of the tumor tissue as measured by 35S-methionine incorporation can be used to indicate the tumor's drug. responsiveness. The extent of viability can also be used to determine an endpoint for maximum responsiveness.

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Abstract

L'invention concerne un procédé d'utilisation d'un système de culture in vitro permettant de mesurer la capacité de prolifération cellulaire ainsi que la viabilité cellulaire de tissus humains, en particulier de tissus tumoraux. L'invention concerne également l'emploi dudit procédé pour évaluer l'efficacité d'un médicament antinéoplastique à inhiber la prolifération ou la viabilité de cellules tumorales.
PCT/US1989/003719 1989-03-20 1989-08-25 PROCEDE ET SYSTEME DE DETERMINATION A L'ETAT NATIF DE LA VIABILITE ET DE LA CAPACITE DE PROLIFERATION DE TISSUS $i(IN VITRO) WO1990011371A1 (fr)

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US32628689A 1989-03-20 1989-03-20
US326,286 1989-03-20

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US5726009A (en) * 1989-03-20 1998-03-10 Anticancer, Inc. Native-state method and system for determining viability and proliferative capacity of tissues in vitro
EP0714448A4 (fr) * 1993-06-29 1998-05-20 Anticancer Inc Systeme et procede de determination, a l'etat natif, de la viabilite et de l'aptitude de proliferation de tissus in vitro
WO1998049343A1 (fr) * 1997-04-29 1998-11-05 North Carolina State University Methodes d'identification de complexes de metaux de transition clivant selectivement les elements regulateurs d'arnm et leurs utilisations
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US7001587B2 (en) 2001-10-24 2006-02-21 The Regents Of The University Of California Measurement of protein synthesis rates in humans and experimental systems by use of isotopically labeled water
US7255850B2 (en) 2002-09-13 2007-08-14 The Regents Of The University Of California Methods for measuring rates of reserve cholesterol transport in vivo, as an index of anti-atherogenesis
US7262020B2 (en) 2003-07-03 2007-08-28 The Regents Of The University Of California Methods for comparing relative flux rates of two or more biological molecules in vivo through a single protocol
USRE40209E1 (en) 1994-06-13 2008-04-01 Matsushita Electric Industrial Co., Ltd. Cell potential measurement apparatus having a plurality of microelectrodes
US7449171B2 (en) 2002-02-12 2008-11-11 The Regents Of The University Of California Measurement of biosynthesis and breakdown rates of biological molecules that are inaccessible or not easily accessible to direct sampling, non-invasively, by label incorporation into metabolic derivatives and catabolic products
US7504233B2 (en) 2002-11-04 2009-03-17 The Regents Of The University Of California Methods for determining the metabolism of sugars and fats in an individual
US8401800B2 (en) 2004-02-20 2013-03-19 The Regents Of The University Of California Molecular flux rates through critical pathways measured by stable isotope labeling in vivo, as biomarkers of drug action and disease activity
US8481478B2 (en) 2002-07-30 2013-07-09 The Regents Of The University Of California Method for automated, large-scale measurement of the molecular flux rates of the proteome or the organeome using mass spectrometry
US8663602B2 (en) 2003-11-25 2014-03-04 The Regents Of The University Of California Method for high-throughput screening of compounds and combinations of compounds for discovery and quantification of actions, particularly unanticipated therapeutic or toxic actions, in biological systems
US9134319B2 (en) 2013-03-15 2015-09-15 The Regents Of The University Of California Method for replacing biomarkers of protein kinetics from tissue samples by biomarkers of protein kinetics from body fluids after isotopic labeling in vivo
US9737260B2 (en) 2011-12-07 2017-08-22 Glaxosmithkline Llc Methods for determining total body skeletal muscle mass
US10386371B2 (en) 2011-09-08 2019-08-20 The Regents Of The University Of California Metabolic flux measurement, imaging and microscopy

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See also references of EP0467885A4 *
The Journal of Histotechnology, Volume 9, Number 1, published in 1986, A. RICHTERS, "Advantages of Combining Histopathology, Tissue Culture, and Ultrastructure in Studying Disease Processes", pages 21-22, see especially the Introduction on page 21. *

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EP0467885A1 (fr) 1992-01-29
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