Patent Application
20210147812
The present application claims the benefit of the Japanese patent applications No. 2017-236374 and No. 2018-195647, which are incorporated herein by reference in their entirety.
The present disclosure includes a method of preparing a cancer spheroid. The present disclosure also includes a method of selecting a colorectal cancer patient for chemotherapy, and a therapeutic method and composition for treating colorectal cancer.
For personalized medicine, patient-derived xenograft (PDX) models prepared by transplanting cancer tissues excised from cancer patients into immunodeficient animals have been studied. The PDX models, however, require repeated transplantation of tumor tissues in order to prepare a sufficient number of model animals for carrying out drug sensitivity tests, and thus take time to prepare. Instead of PDX, spheroids prepared from cells of patient tissues have been considered. However, spheroids have not been successfully prepared with sufficient efficiency for clinical application.
Regarding production of tissue-derived spheroids, it has been reported that spheroids are prepared by culturing human colonic epithelial cells in a conditioned medium of L-WRN fibroblasts that produce Wnt3a, R-spondin-3 and Noggin, which are essential for proliferation of tissue stem cells, and a ROCK inhibitor Y27632 and a TGF-β type I receptor inhibitor SB431542. It has also been reported that spheroids are prepared by culturing tumor cells of a colorectal cancer model mouse in a basic medium supplemented with Y27632 and SB431542.
Colorectal cancer is one of the leading causes of cancer death worldwide. For example, in the US, the 5-year survival rate for patients with stage IV colorectal cancer is only 13%. This poor prognosis is due to the difficulty in selecting an appropriate chemotherapy for individual patients. The current selection criteria for EGFR-targeted therapies in colorectal cancer are the absence of activating mutations in RAS pathway genes in tumor DNA. However, in clinical, 40-50% of selected patients do not respond to anti-EGFR therapies.
Genomic sequences and gene expression information have also been used to predict response to chemotherapy. However, it is still difficult to identify reliable biomarkers or gene expression information due to the genetic or epigenetic complexity of patients with colorectal cancer.
The fibroblast growth factor (FGF) family is one of the factors that directly promote the growth of epithelial cells. The FGF signal is considered to play an important role in cancer cell proliferation, migration, and survival, and FGF and its receptor family are expected as therapeutic targets leading to new drug development. Fibroblast growth factor receptor (FGFR) family proteins are receptor tyrosine kinases and the FGFR family is composed of four types of receptors, FGFR1 to 4. Homeostatic activation of the FGFR signaling pathway is thought to be deeply involved in the growth of some cancer cells. However, there is no reliable method to assess the sensitivity of colorectal cancer patients for adaptation to chemotherapy that inhibits the FGF signal.
An object of the present disclosure is to provide an efficient method to prepare a cancer spheroid.
Another object of the present disclosure is to provide a method of selecting a colorectal cancer patient who is adaptive to an FGFR inhibitor. A further object of the present disclosure is to provide a therapeutic method and composition for treating colorectal cancer.
In an aspect, the present disclosure relates to a method of preparing a cancer spheroid, comprising
culturing a cell population obtained from a cancer tissue of a patient in a medium containing
a ROCK inhibitor, a TGF-β type I receptor inhibitor, EGF and bFGF; and
an agent selected from an adenosine receptor agonist, a PPAR agonist, a calcium-activated potassium-channel (KCa) activator, an NMDA-type glutamate receptor agonist, a histamine H3 receptor agonist, and a retinoic acid receptor (RXR) β2 agonist.
In a further aspect, the present disclosure relates to a method of evaluating drug sensitivity of a patient, or a method of screening a therapeutic agent for a cancer, comprising
preparing a cancer spheroid by the method as described above, and
contacting the cancer spheroid thus obtained or a cancer spheroid derived therefrom with a candidate agent in vitro.
In a further aspect, the present disclosure relates to a method of preparing a patient-derived spheroid xenograft (PDSX) model, comprising
preparing a cancer spheroid by the method as described above, and
transplanting the cancer spheroid thus obtained or a cancer spheroid derived therefrom to a non-human animal.
In a further aspect, the present disclosure relates to a method of evaluating drug sensitivity of a patient, or a method of screening a therapeutic agent for a cancer, comprising
preparing a PDSX model by the method as described above, and
administering a candidate agent to the PDSX model thus obtained.
In a further aspect, the present disclosure relates to a method of detecting microsatellite instability (MSI), comprising
preparing a cancer spheroid by the method as described above, and
determining MSI status or a mutation of a target gene of a mismatch repair gene in the cancer spheroid thus obtained or a cancer spheroid derived therefrom.
In a further aspect, the present disclosure relates to a method of selecting a colorectal cancer patient being sensitive to an FGFR inhibitor, comprising
treating a cancer spheroid or a patient-derived spheroid xenograft (PDSX) model derived from a colorectal cancer patient with an FGFR inhibitor, and
determining response to the FGFR inhibitor of the cancer spheroid or the PDSX model, and
selecting the colorectal cancer patient when the response to the FGFR inhibitor is positive.
In a further aspect, the present disclosure relates to a pharmaceutical composition for treating colorectal cancer comprising an FGFR inhibitor.
The present disclosure provides an efficient method for preparing a cancer spheroid, and enables an efficient drug sensitivity test for personalized medicine and an efficient drug screening for drug development, and also enables a highly sensitive MSI test.
The present disclosure also provides a method for selecting a colorectal cancer patient being sensitive to an FGFR inhibitor, and therapeutic method and composition for treating colorectal cancer using an FGFR inhibitor.
Unless otherwise defined, the terms used herein are read as generally understood by a skilled person in the technical fields such as organic chemistry, medical sciences, pharmaceutical sciences, molecular biology, and microbiology. Several terms used herein are defined as described below. The definitions herein take precedence over the general understanding.
When a numerical value is accompanied with the term “about”, the value is intended to represent any value in the range of −10% of the value to +10% of the value. A range defined with a value of the lower limit and a value of the upper limit covers all values from the lower limit to the upper limit, including the values of both limits. When a range is accompanied with the term “about”, both limits are read as accompanied with the term. For example, “about 20 to 30” is read as “20±10% to 30±10%.”
As used herein, a cancer spheroid means a cell mass containing a tumor initiating cell. The tumor initiating cell means a cell being able to form a tumor when transplanted into an immunodeficient animal. In general, a cancer spheroid contains about 100 cells to about 10,000 cells, preferably about 500 cells to about 2000 cells, and has a shape being close to a sphere with a diameter of about 0.01 mm to about 2 mm, preferably about 0.1 mm to about 0.5 mm. Here, the “diameter” means the length of the longest axis of a cell mass.
The “cell population obtained from a cancer tissue of a patient” as used herein can be prepared by a conventional method. For example, the methods described in Miyoshi H, Stappenbeck T S. Nat Protoc. 2013; 8: 2471-82 or VanDussen K L, et al., Gut. 2015; 64: 911-20 may be used. In an embodiment, the cell population obtained from a cancer tissue of a patient is a cell population obtained by subjecting a cancer tissue obtained from a patient or an epithelial tissue separated from such a cancer tissue to an enzyme treatment (for example, collagenase treatment). The cell population obtained from a cancer tissue of a patient comprises a cancer cell and a tumor initiating cell.
In general, cells are obtained from a cancer tissue in a volume of about 0.1 cm3 to 5.0 cm3, about 0.2 cm3 to 3.0 cm3, or about 0.5 cm3 to 2.0 cm3, preferably about 0.5 cm3 to 1.0 cm3, and cultured. The cells are preferably cultured with a basement membrane matrix such as Matrigel® (Corning) or Cultrex® Basement Membrane Extract (Trevigen). For example, 103 cells to 105 cells are mixed with a basement membrane matrix, and the cell suspension thus obtained is seeded on a culture vessel (such as culture plate or culture dish). After the basement membrane matrix is solidified at 37° C., a culture solution is overlaid to start cell culture.
In an aspect, the present disclosure relates to a method of preparing a cancer spheroid, comprising
culturing a cell population obtained from a cancer tissue of a patient in a medium containing
a ROCK inhibitor, a TGF-β type I receptor inhibitor, EGF and bFGF; and
an agent selected from an adenosine receptor agonist, a PPAR agonist, a calcium-activated potassium-channel (KCa) activator, an NMDA-type glutamate receptor agonist, a histamine H3 receptor agonist, and a retinoic acid receptor (RXR) β2 agonist.
Examples of cancers include colorectal cancer, gastric cancer, prostate cancer, breast cancer, cervical cancer, ovarian cancer, lung cancer, hepatocellular cancer, kidney cancer, pancreatic cancer, and bladder cancer. Preferably, the cancer is colorectal cancer. The cancer tissue may be a tissue obtained from a primary lesion or a metastatic lesion, but is preferably a tissue obtained from a primary lesion. The cancer tissue may be a surgical specimen or a biopsy specimen.
The medium may be any medium commonly used for spheroid culture, and examples thereof include Advanced DMEM/F-12 (Thermo Fisher) and DMEM/F12 (Thermo Fisher) supplemented with ITS supplement (Thermo Fisher).
Examples of adenosine receptor agonists include NECA (5′-(N-ethyl-carboxamido)-adenosine), CV1808 (2-phenylaminoadenosine), and BAY 60-6583 (2-[[6-Amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]-2-pyridinyl]thio]-acetamide). Preferably, the adenosine receptor agonist is NECA or CV1808, and more preferably NECA. The adenosine receptor agonist may be used, for example, at a concentration of 0.001 μM to 10 μM, preferably 0.1 μM to 5 μM, more preferably 0.1 μM to 1 μM.
The PPAR agonist includes a PPARα agonist, a PPARγ agonist and a PPARδ agonist.
Examples of PPARα agonists include GW7647 (2-methyl-2-[[4-[2-[[(cyclohexylamino)carbonyl](4-cyclohexylbutyl)amino]ethyl]phenyl]thio]-propanoic acid) and CP 775146 (2-Methyl-2-[3-[(3S)-1-[2-[4-(1-methylethyl)phenyl]acetyl]-3-piperidinyl]phenoxy]-propanoic acid);
Examples of PPARγ agonists include 526948 ([[4-[2-(6-Benzoyl-2-oxo-3(2H)-benzothiazolyl)ethoxy]phenyl]methyl]-1,3-propanedioic acid dimethyl ester) and Troglitazone (5-[[4-[(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy]phenyl]methyl]-2,4-thiazolidinedione);
Examples of PPARδ agonists include GW0742 ([4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methyl phenoxy]-acetic acid), L-165,041 ([4-[3-(4-Acetyl-3-hydroxy-2-propylphenoxy)propoxy]phenoxy]acetic acid), and GW 501516 (2-[2-Methyl-4-[[[4-methyl-2-[4-(trifluoromethyl)phenyl]-5-thiazolyl]methyl]thio]phenoxy]acetic acid).
Examples of KCa activators include DCEBIO (5,6-Dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one), SKA-31 (Naphtho[1,2-d]thiazol-2-ylamine), and 1-EBIO (1-Ethyl-2-benzimidazolinone). Preferably, the KCa activator is DCEBIO or SKA-31. The KCa activator may be used, for example, at a concentration of 0.1 μM to 50 μM, preferably 1 μM to 30 μM, more preferably about 10 μM.
Examples of NMDA-type glutamate receptor agonists include NMDA (N-Methyl-D-aspartic acid) and Homoquinolinic acid. Preferably, the NMDA-type glutamate receptor agonist is NMDA. The NMDA-type glutamate receptor agonist may be used at a concentration of, for example, 0.1 μM to 50 μM, preferably 1 μM to 30 μM, more preferably about 10 μM.
Examples of histamine H3 receptor agonists include Immepip (4-(1H-Imidazol-4-ylmethyl)piperidine) and Immethridine (4-(1H-Imidazol-4-ylmethyl)pyridine). Preferably, the histamine H3 receptor agonist is Immepip. The histamine H3 receptor agonist may be used at a concentration of, for example, 0.1 μM to 50 μM, preferably 1 μM to 30 μM, more preferably about 10 μM.
Examples of RXRβ2 agonists include AC55649 (4′-Octyl-[1,1′-biphenyl]-4-carboxylic acid) and AC 261066 (4-[4-(2-Butoxyethoxy-)-5-methyl-2-thiazolyl]-2-fluorobenzoic acid). Preferably, the RXRβ2 agonist is AC55649. The RXRβ2 agonist may be used, for example, at a concentration of 0.1 μM to 50 μM, preferably 1 μM to 30 μM, more preferably about 10 μM.
Examples of ROCK inhibitors include Y27632 ((R)-(+)-trans-4-(1-Aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide) and H1152 ((S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine). Preferably the ROCK inhibitor is Y27632. The ROCK inhibitor may be used, for example, at a concentration of 0.1 μM to 50 μM, preferably 1 μM to 30 μM, more preferably about 10 μM.
Examples of TGF-β (transforming growth factor-(3) type I receptor inhibitors include SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide) and A83-01 (3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide). Preferably, the TGF-β type I receptor inhibitor is SB431542. The TGF-β type I receptor inhibitor may be used, for example, at a concentration of 0.01 μM to 10 μM, preferably 0.1 μM to 10 μM, more preferably 0.5 μM to 10 μM.
EGF (epidermal growth factor) and bFGF (basic fibroblast growth factor) may be a recombinant human protein. EGF may be used at a concentration of, for example, 10 ng/ml to 100 ng/ml, preferably about 50 ng/ml. bFGF may be used at a concentration of, for example, 50 ng/ml to 200 ng/ml, preferably about 100 ng/ml.
The medium usually contains serum (such as fetal bovine serum (FBS)). The serum may be used at a concentration of about 1-10%, preferably about 5%.
The medium may contain two or more agents selected from an adenosine receptor agonist, a PPAR agonist, a KCa activator, an NMDA-type glutamate receptor agonist, a histamine H3 receptor agonist, and a RXR 132 agonist.
The medium may further contain an antibiotic such as penicillin, streptomycin, or plasmin, L-glutamine, or B27 supplement (Thermo Fisher).
The period of culturing cells is not particularly limited to a specific period, and the culture may be continued until an appropriate number of cancer spheroid cells are obtained. In general, the culture is continued until about 1×105 to about 1×107, preferably about 106 cancer spheroid cells are obtained. The culture period may be, for example, one week to two months (for example, one week to four weeks, or one month to two months). During the culture period, the medium is preferably replaced with a fresh medium every other day or every few days (for example, two or three days). Usually, during the continuous culture, a procedure of treating the formed cell mass with an enzyme (such as trypsin) to separate cells and re-forming a cell mass is performed once or more.
The cancer spheroid thus obtained may be further cultured for several days to several weeks until the number of cells becomes an appropriate number depending on the purpose of use. Also, the cancer spheroid may be stored frozen until use, and may be cultured until an appropriate cell number is obtained after thawing. The cancer spheroid may be passaged during the culture. As used herein, a cancer spheroid derived from the cancer spheroid thus obtained means a cancer spheroid obtained by passaging a cancer spheroid prepared by the method of the present disclosure. The passaging may or may not involve treating the cancer spheroid with an enzyme (such as trypsin) to separate cells and re-forming a cancer spheroid.
A cancer spheroid may be provided as a composition comprising the cancer spheroid and a solution suitable for culturing or storing the cancer spheroid. The composition comprising a cancer spheroid may be frozen, in which case the composition may comprise a cancer spheroid and a suitable cryopreservation liquid.
A cancer spheroid can be used, for example, for in vivo or in vitro drug sensitivity evaluation, drug screening, and microsatellite instability (MSI) test. A cancer spheroid can also be used for searching for a biomarker of drug sensitivity based on the results of drug sensitivity evaluation. The results of drug sensitivity evaluation can be used not only for selection of treatment for patients, but also for selection of clinical trial participants in new drug development.
A reporter gene may be introduced into a cell in a cancer spheroid to monitor cell proliferation. Examples of reporter genes include luciferase and green fluorescent protein (GFP). The reporter gene can be introduced using a vector such as a plasmid vector or a viral vector. In an embodiment, the reporter gene is introduced into a cell in a cancer spheroid by a viral vector such as a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector, preferably by a lentiviral vector. The vector can be introduced by a conventional method, for example, the methods described in the example section of the present disclosure or Miyoshi H, Stappenbeck T S. Nat Protoc. 2013; 8: 2471-82. Introduction of the reporter gene is preferably performed after a cancer spheroid is treated with an enzyme (such as trypsin) to separate cells. After the reporter gene is introduced, a spheroid can be re-formed and used for an assay.
A patient-derived spheroid xenograft (PDSX) model can be prepared by transplanting a cancer spheroid into a non-human animal. Examples of non-human animals include immunodeficient animals such as nude mice, nude rats, SCID mice, NOD-SCID mice, and NOG mice. The transplantation site is not particularly limited to a specific site, but subcutaneous transplantation is preferred. The number of cells in a cancer spheroid to be transplanted may be any number, but in the case of a mouse, it is usually about 1×105 to about 9×105, preferably about 5×105. It is preferable that the non-human animal is used for a test when the tumor volume reaches about 300-500 mm3 after the transplantation.
By administering a candidate agent to a PDSX model and determining response to the candidate agent, drug sensitivity of a patient from whom a cancer spheroid is derived can be evaluated, or drug screening can be carried out. The response of cells can be determined based on the volume or a histological feature of a tumor tissue. Examples of histological features include the number of mitotic figures and the number of degenerated cells. For example, when an increase in the volume of a tumor tissue is significantly suppressed, or when a histological feature is improved (for example, when the number of mitotic figures is decreased or the number of degenerated cells is increased) as compared to a control PDSX model, to which the candidate agent has not been administered, the patient from whom the cancer spheroid is derived is evaluated as being sensitive to the candidate agent, or the candidate agent is evaluated as being effective in treating cancer.
Examples of candidate agents include, but not limited to, single compounds such as low molecular weight compounds, proteins, antibodies, peptides, nucleic acids; libraries of compounds, antibodies, nucleic acids, or other components; or cell extracts, cell culture supernatants, microorganism fermentation products, marine organism extracts, and plant extracts. When drug sensitivity of a patient is evaluated, the candidate agent may be a known drug for cancer therapy.
Drug sensitivity evaluation and drug screening can also be carried out by contacting a cancer spheroid with a candidate agent in vitro and determining response to the candidate agent. For example, when the candidate agent significantly suppresses an increase in the volume of a cancer spheroid or improves a histological feature of a cancer spheroid compared to a control cancer spheroid, which has not been contacted with the candidate agent, the patient is evaluated as being sensitive to the candidate agent, or the candidate agent is evaluated as being effective in treating cancer.
MSI is a phenomenon in which a microsatellite shows a different number of repeats in a tumor tissue as compared to a non-tumor tissue due to dysfunction of a mismatch repair gene. The MSI test is known to be useful as an adjunctive diagnosis of Lynch syndrome, which is caused by germline mutations of mismatch repair genes, or in predicting effects of an immune checkpoint inhibitor (such as anti-PD-1 antibody) on colorectal cancer. The MSI test usually examines the number of microsatellite repeats in a tumor tissue and a non-tumor tissue, and a cancer spheroid can be used as a tumor tissue in place of a surgical or biopsy specimen. The use of a cancer spheroid enables more accurate examination.
The number of microsatellite repeats can be determined by a conventional method. For example, a region containing a microsatellite repeat sequence is amplified by PCR from DNA obtained from a cancer spheroid and a non-tumor tissue (or blood when it is difficult to collect a non-tumor tissue), and the number of repeats of the microsatellite is compared. The result is MSI-positive when the number of microsatellite repeats in the cancer spheroid is changed compared to the non-tumor tissue. The MSI status is usually determined using five microsatellite markers of BAT25, BAT26, D5S346, D2S123, and D17S250 (Bethesda Panel). The MSI status is determined as MSI-H (MSI-high) when two or more of the markers show MSI, as MSI-L (MSI-low) when one of the markers shows MSI, and as MSS (microsatellite stable) when MSI was not observed. When one or more additional markers are used in addition to the five makers, the MSI status is determined as MSI-H when 30-40% or more of the markers used shows MSI, and as MSI-L when less than 30% of the markers does.
The MSI test can also be carried out by examining a mutation in a target gene of a mismatch repair gene, which is a gene showing abnormality due to functional abnormality of the mismatch repair gene. Examples of target genes include TGFBR2, IGF2R, BAX, and CASP5. The mutation can be examined by using DNA obtained from a cancer spheroid, and a conventional method for detecting a gene mutation (for example, a nucleic acid hybridization method using a DNA chip or a DNA microarray, direct sequence method, TaqMan PCR method, RFLP method, or PCR-SSCP method). DNA obtained from a cancer spheroid has a higher purity than that from a surgical or biopsy specimen and enables detection of a heterozygous mutation that cannot be detected previously, and thus enables an accurate examination.
In an aspect, the present disclosure provides a method of selecting a colorectal cancer patient being sensitive to an FGFR inhibitor, comprising treating a cancer spheroid or a patient-derived spheroid xenograft (PDSX) model derived from a colorectal cancer patient with an FGFR inhibitor, and determining response to the FGFR inhibitor of the cancer spheroid or the PDSX model, and selecting the colorectal cancer patient when the response to the FGFR inhibitor is positive.
The large intestine is divided into the colon and the rectum, and the term “colorectal cancer” in the present disclosure includes colon cancer and rectal cancer. Colorectal cancer is divided into stages 0, I, II, III, and IV according to the degree of progression, and the term “colorectal cancer” in the present disclosure includes any stage of cancer.
As used herein, the term “fibroblast growth factor receptor (FGFR) inhibitor” means a substance that inhibits signal transduction mediated by FGFR. There are four types of FGFR, FGFR1, FGFR2, FGFR3, and FGFR4, and an FGFR inhibitor can inhibit one or more of these four types of FGFR. In an embodiment, the FGFR inhibitor inhibits three types of FGFR1, FGFR2, and FGFR3, or four types of FGFR1, FGFR2, FGFR3, and FGFR4. The FGFR inhibitor can be a small molecule compound, a protein, an antibody, a peptide, or a nucleic acid. Examples of FGFR inhibitors include erdafitinib (JNJ42756493), rogolatinib (BAY1163877), AZD4547, BGJ398, BLU554, LY2874455, ASP5878 and ARQ087. In an embodiment, the FGFR inhibitor is erdafitinib.
As used herein, the term “cancer spheroid derived from a colorectal cancer patient” includes a cancer spheroid prepared from a cell population obtained from a cancer tissue of a colorectal cancer patient, and a cancer spheroid obtained by passaging such a cancer spheroid. The cancer tissue may be a tissue obtained from a primary lesion or a metastatic lesion, but is preferably a tissue obtained from a primary lesion. The cancer tissue can be a surgical specimen or a biopsy specimen. As used herein, “a PDSX model derived from a colorectal cancer patient” is a model animal prepared by transplanting a cancer spheroid derived from a colorectal cancer patient into a non-human animal.
In this aspect, the cancer spheroid may be prepared by any method from a cell population obtained from a cancer tissue of a patient. For example, the methods described in Non-Patent Documents 1 to 7 of the present disclosure can be used.
In an embodiment, the cancer spheroid is prepared by a method that comprises culturing a cell population obtained from a cancer tissue of a patient in a medium containing a ROCK inhibitor, a TGF-β type I receptor inhibitor, EGF and bFGF. In this embodiment, the cancer spheroid can be prepared according to the descriptions of item 2 above, except that the medium in this embodiment does not necessarily contain an agent selected from an adenosine receptor agonist, a PPAR agonist, a KCa activator, an NMDA-type glutamate receptor agonist, a histamine H3 receptor agonist, and a RXR β2 agonist.
In a further embodiment, the cancer spheroid is prepared by the method for preparing a cancer spheroid of the present disclosure as described in item 2 above. The method for preparing a cancer spheroid of the present disclosure enables efficient preparation of a cancer spheroid.
By treating a cancer spheroid or PDSX model derived from a colorectal cancer patient with an FGFR inhibitor and determining response of the cancer spheroid or PDSX model to the FGFR inhibitor, sensitivity of the colorectal cancer patient to the FGFR inhibitor can be evaluated. When the response to the FGFR inhibitor is positive, the patient is evaluated as sensitive to the FGFR inhibitor and can be selected for the treatment with the FGFR inhibitor.
In the present disclosure, a cancer spheroid is treated with an FGFR inhibitor in vitro. An FGFR inhibitor is typically added to the culture medium of the cancer spheroid. A cancer spheroid is preferably cultured with a basement membrane matrix such as Matrigel® or Cultrex® Basement Membrane Extract. For example, a cancer spheroid derived from a colorectal cancer patient is treated with an enzyme (such as trypsin) to separate cells, mixed with a basement membrane matrix at about 5×104 to 2×106 cells/ml, and the resulting cell suspension is seeded on a culture vessel (such as culture plate or culture dish). After the basement membrane matrix is solidified at 37° C., a medium is overlaid for culture. The medium may be any medium commonly used for spheroid culture as described in item 2 above. An FGFR inhibitor is added at a concentration that is appropriately determined depending on the FGFR inhibitor to be evaluated. For example, an FGFR inhibitor may be added at final concentration of 10−1° M to 10−5 M or 10−9 M to 10−6 M. After a period of time from the start of the treatment with the FGFR inhibitor, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days later, the response to the FGFR inhibitor is determined.
In the present disclosure, the PDSX model is a non-human animal. Examples of non-human animals for preparing a PDSX model include immunodeficient animals such as nude mice, nude rats, SCID mice, NOD-SCID mice and NOG mice. The transplantation site is not particularly limited to a specific site, but subcutaneous transplantation is preferred. The number of cells in a cancer spheroid to be transplanted may be any number, but in the case of a mouse, it is usually about 1×105 to about 9×105, preferably about 5×105. It is preferable that the non-human animal is used for a test when the tumor volume reaches about 300-500 mm3 after the transplantation. An FGFR inhibitor is administered at a dose appropriately determined according to the FGFR inhibitor to be evaluated. For example, an FGFR inhibitor is administered at 0.1 mg/kg body weight to 100 mg/kg body weight, 1 mg/kg body weight to 30 mg/kg body weight, or 10 mg/kg to 30 mg/kg body weight. An FGFR inhibitor is administered at an appropriately determined administration interval, and may be administered once or multiple times a day, which may be performed every day or every few days, or every week or every few weeks. The response to the FGFR inhibitor is determined after a period of time, for example one week to several months, for example one week, two weeks, three weeks or four weeks after the start of the treatment with the FGFR inhibitor.
The response of a cancer spheroid to an FGFR inhibitor can be determined based on, for example, growth or a histological feature of a cancer spheroid. The growth of a cancer spheroid can be determined by measuring a signal (for example, fluorescence) from a reporter gene contained in a cell of the cancer spheroid. The response of a PDSX model to an FGFR inhibitor can be determined based on the volume or a histological feature of a tumor tissue that is derived from the transplanted cancer spheroid. The volume of a tumor tissue can be calculated by the following formula: tumor volume (mm3)=[length (mm)×width2 (mm2)]/2 (wherein the length means the longer tumor axis and the width means the shorter tumor axis). The histological feature of a cancer spheroid or a tumor tissue can be examined by hematoxylin/eosin staining (H&E staining) or immunostaining for a cell proliferation marker (such as Ki67, PCNA, or MCM2). Histological features include the number of mitotic figures and the number of degenerated cells.
The response to an FGFR inhibitor is determined by comparing a value obtained from a cancer spheroid or PDSX model treated with the FGFR inhibitor with a reference value. In an embodiment, the response to an FGFR inhibitor is determined to be positive when the value obtained from a cancer spheroid or PDSX model treated with the FGFR inhibitor is lower than a reference value. The reference value may be a value obtained from a cancer spheroid or PDSX model that has not been treated with the FGFR inhibitor (control value). Alternatively, the reference value may be 90% to 10% (such as 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%) of the control value, for example 90% to 50% of the control value. For example, when the growth of the cancer spheroid treated with an FGFR inhibitor, the volume of a tumor tissue of the PDSX model treated with an FGFR inhibitor, or the number of mitotic figures in the cancer spheroid or PDSX model is lower than the reference value, the response to the FGFR inhibitor is determined to be positive.
In a different embodiment, the response to an FGFR inhibitor is determined to be positive when the value obtained from a cancer spheroid or PDSX model treated with the FGFR inhibitor is higher than a reference value. The reference value may be the control value. Alternatively, the reference value may be 110-200% (such as 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%) of the control value, for example 110%-150% of the control value. For example, when the number of degenerated cells in the cancer spheroid or PDSX model treated with a FGFR inhibitor is higher than the reference value, the response to the FGFR inhibitor is determined to be positive.
The comparison with a reference value may or may not include a statistical analysis. Examples of statistical analysis methods include one-way analysis of variance (one-way ANOVA), two-way analysis of variance (two-way ANOVA), Dunnett's test, and Tukey's test.
In an embodiment, the response of a cancer spheroid to an FGFR inhibitor is determined based on the growth of the cancer spheroid, and the reference value to be used is 90%-50%, for example 70%, of the control value. When the growth of the cancer spheroid treated with an FGFR inhibitor is lower than the reference value, the response to the FGFR inhibitor is determined to be positive.
In a different embodiment, the response to an FGFR inhibitor of a PDSX model is determined based on the volume of a tumor tissue of the PDSX model, and the reference value to be used is the control value. When the volume of a tumor tissue of the PDSX model treated with an FGFR inhibitor is statistically significantly lower than the reference value, the response to the FGFR inhibitor is determined to be positive
As shown in the example section, it was not possible to select a colorectal cancer patient being sensitive to an FGFR inhibitor based on the copy number or mRNA amount of the FGFR gene, which has been used as a patient selection criterion for an FGFR inhibitor. The patient selection method of the present disclosure enables selection of a colorectal cancer patient who is sensitive to an FGFR inhibitor, and expands the application of FGFR inhibitors.
The method of the present disclosure may include, in addition to the response to an FGFR inhibitor, determining the response to a combination of the FGFR inhibitor and one or more agents different from the FGFR inhibitor. When the one or more agents enhance the response to the FGFR inhibitor, the agent(s) can be used with the FGFR inhibitor in the treatment of colorectal cancer. The one or more agents may be those exemplified herein as agents that may be used in combination with an FGFR inhibitor in the treatment of colorectal cancer.
The treatment of colorectal cancer may be treatment before or after resection of a cancer tissue. For example, the treatment of colorectal cancer can be treatment of unresectable colorectal cancer or treatment to prevent recurrence after resection of a cancer tissue. For the treatment of colorectal cancer, an effective amount of an FGFR inhibitor is administered to a colorectal cancer patient. As used herein, “an effective amount” is an amount capable of exerting a desired effect in the treatment of colorectal cancer, such as elimination or shrinkage of tumor, suppression of tumor growth, or prevention of recurrence. An FGFR inhibitor is administered at a dose appropriately determined according to the age or body weight of the patient, severity of the disease, concomitant drug or other factors, and may be administered, for example, at 0.01 mg/kg body weight to 100 mg/kg body weight, 0.1 mg/kg body weight to 100 mg/kg body weight, 0.1 mg/kg body weight to 10 mg/kg body weight, or 0.1 mg/kg body weight to 1 mg/kg body weight per day. In an embodiment, the FGFR inhibitor is erdafitinib and administered at 1 mg/day to 30 mg/day, 1 mg/day to 15 mg/day, 1 mg/day to 12 mg/day, or 1 mg/day to 10 mg/day. An FGFR inhibitor can be administered orally or parenterally (for example, by intravenous administration, intramuscular administration, or subcutaneous administration). An FGFR inhibitor may be administered once, twice, three times, four times or more times a day, and may be administered daily or at regular intervals.
An FGFR inhibitor may be combined with endoscopic resection, surgical resection, or radiation therapy for the treatment of colorectal cancer, or may be combined with one or more agents in chemotherapy. The one or more agents can be selected from, for example, anti-cancer agents such as 5-fluorouracil (5-FU), tegafur, tegafur-uracil (UFT), doxyfluridine (5′-DFUR), carmofur, tegafur-guimeracil-oteracil potassium (S-1), capecitabine (Cape), regorafenib, trifluridine-tipiracil hydrochloride (TAS-102), mitomycin C, irinotecan (IRI), and oxaliplatin (OX), and molecular targeting agents such as vascular endothelial growth factor (VEGF) inhibitors (such as bevacizumab and aflibercept), vascular endothelial growth factor receptor (VEGFR) inhibitors (such as ramucirumab), EGFR inhibitors (such as cetuximab, panitumumab, or erlotinib). In an embodiment, an FGFR inhibitor is combined with an EGFR inhibitor. In an embodiment, the EGFR inhibitor is cetuximab or panitumumab. In a further embodiment, the EGFR inhibitor is cetuximab. When it is described herein that an FGFR inhibitor is used in combination with one or more agents, it also includes the case where these are used in combination with one or more additional agents.
In an embodiment, an FGFR inhibitor is used in combination with a chemotherapy such as FOLFOX therapy (drip 5-FU+levofolinate+OX), FOLFIRI therapy (5-FU+levofolinate+IRI), FOLFOXIRI therapy (5-FU+levofolinate+OX+IRI), CapeOX therapy (Cape+OX), SOX therapy (S-1+OX), or IRIS therapy (S-1+IRI), or a combination of such a chemotherapy and one or more molecular targeting agents. In an embodiment, the one or more molecular targeting agents include an EGFR inhibitor. In an embodiment, the EGFR inhibitor is cetuximab or panitumumab. In a further embodiment, the EGFR inhibitor is cetuximab.
When two or more agents are used in combination, some or all of these agents may be comprised in one composition, or all agents may be comprised in separate compositions. In addition, the administration schedule of these agents may be the same or different.
The pharmaceutical composition comprising an FGFR inhibitor can be formulated by a conventional method. The pharmaceutical composition may be in the form of tablets, capsules, powders, granules, suspensions, syrups, elixirs, or injections. Depending on the dosage form, the pharmaceutical composition may include, in addition to the FGFR inhibitor, a pharmaceutically acceptable additive selected from, for example, excipients, disintegrants, binders, lubricants, stabilizers, buffers, surfactants, preservatives, and sweeteners, and/or a pharmaceutically acceptable carrier selected from, for example, water, saline, oils, and alcohols.
Examples of embodiments of the present disclosure are provided below.
1. A method of preparing a cancer spheroid, comprising culturing a cell population obtained from a cancer tissue of a patient in a medium containing
a ROCK inhibitor, a TGF-β type I receptor inhibitor, EGF and bFGF; and
an agent selected from an adenosine receptor agonist, a PPAR agonist, a calcium-activated potassium-channel (KCa) activator, an NMDA-type glutamate receptor agonist, a histamine H3 receptor agonist, and a retinoic acid receptor (RXR) β2 agonist.
2. The method of item 1, wherein the cancer is colorectal cancer.
3. The method of item 1 or 2, wherein the cancer tissue is a tissue obtained from a primary lesion.
4. The method of any one of items 1-3, wherein the agent is an adenosine receptor agonist.
5. The method of item 4, wherein the adenosine receptor agonist is NECA or CV1808.
6. The method of item 5, wherein the adenosine receptor agonist is NECA.
7. The method of any one of items 1-3, wherein the agent is a PPAR agonist.
8. The method of item 7, wherein the PPAR agonist is GW0742, GW7647, 526948 or L-165,041.
9. The method of any one of items 1-3, wherein the agent is a KCa activator.
10. The method of item 9, wherein the KCa activator is DCEBIO or SKA-31.
11. The method of any one of items 1-3, wherein the agent is an NMDA-type glutamate receptor agonist.
12. The method of item 11, wherein the NMDA-type glutamate receptor agonist is NMDA.
13. The method of any one of items 1-3, wherein the agent is a histamine H3 receptor agonist.
14. The method of item 13, wherein the histamine H3 receptor agonist is imepip.
15. The method of any one of items 1-3, wherein the agent is a RXR β2 agonist.
16. The method of item 15, wherein the RXR β2 agonist is AC55649.
17. The method of any one of items 1-3, wherein the agent is selected from NECA, CV1808, GW0742, GW7647, S26948, L-165,041, DCEBIO, SKA-31, NMDA, imepip and AC55649.
18. The method of any one of items 1-17, wherein the ROCK inhibitor is Y27632.
19. The method of any one of items 1-18, wherein the TGF-β type I receptor inhibitor is SB431542.
20. The method of any one of items 1-19, wherein the medium further comprises serum.
21. The method of any one of items 1-20, wherein the cell population is cultured for one week to two months.
22. A composition comprising a cancer spheroid obtained by the method of any one of items 1-21 or a cancer spheroid derived therefrom.
23. A method of evaluating drug sensitivity of a patient, comprising
preparing a cancer spheroid by the method of any one of items 1-21, and
contacting the cancer spheroid thus obtained or a cancer spheroid derived therefrom with a candidate agent in vitro.
24. A method of screening a therapeutic agent for a cancer, comprising
preparing a cancer spheroid by the method of any one of items 1-21, and
contacting the cancer spheroid thus obtained or a cancer spheroid derived therefrom with a candidate agent in vitro.
25. The method of item 23 or 24, wherein the method further comprises introducing a reporter gene into a cell within the cancer spheroid.
26. A method of preparing a patient-derived spheroid xenograft (PDSX) model, comprising
preparing a cancer spheroid by the method of any one of items 1-21, and
transplanting the cancer spheroid thus obtained or a cancer spheroid derived therefrom to a non-human animal.
27. A method of evaluating drug sensitivity of a patient, comprising
preparing a PDSX model by the method of item 26, and
administering a candidate agent to the PDSX model thus obtained.
28. A method of screening a therapeutic agent for a cancer, comprising
preparing a PDSX model by the method of item 26, and
administering a candidate agent to the PDSX model thus obtained.
29. A method of detecting microsatellite instability (MSI), comprising
preparing a cancer spheroid by the method of any one of items 1-21, and
determining MSI status or a mutation of a target gene of a mismatch repair gene in the cancer spheroid thus obtained or a cancer spheroid derived therefrom.
30. The method of item 29, wherein the method is for predicting efficacy of an immune checkpoint inhibitor.
31. The method of item 30, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.
32. The method of any one of items 29-31, wherein the determining comprises determining the number of repeats for each of microsatellite markers BAT25, BAT26, D5S346, D2S123, and D17S250.
33. The method of any one of items 29-31, wherein the target gene of a mismatch repair gene is TGFBR2, IGF2R, BAX, or CASP5.
Further examples of embodiments of the present disclosure are provided below.
1′. A method of selecting a colorectal cancer patient being sensitive to an FGFR inhibitor, comprising treating a cancer spheroid or a patient-derived spheroid xenograft (PDSX) model derived from a colorectal cancer patient with an FGFR inhibitor, and determining response to the FGFR inhibitor of the cancer spheroid or the PDSX model, and selecting the colorectal cancer patient when the response to the FGFR inhibitor is positive.
2′. The method of item 1′, wherein the treating comprises treating a cancer spheroid derived from a colorectal cancer patient with an FGFR inhibitor.
3′. The method of item 2′, wherein the response to the FGFR inhibitor is determined based on growth or a histological feature of the cancer spheroid.
4′ The method of item 1′, wherein the treating comprises treating a PDSX model with an FGFR inhibitor.
5′ The method of item 4′, wherein the response to the FGFR inhibitor is determined based on the volume or a histological feature of a tumor tissue of the PDSX model.
6′. The method of any one of items 1′-5′, wherein the method further comprises preparing the cancer spheroid from a cell population obtained from a cancer tissue of the colorectal cancer patient.
7′. The method of any one of items 1′-6′, wherein the method further comprises preparing the PDXS model by transplanting a cancer spheroid derived from the colorectal cancer patient to a non-human animal.
8′. The method of any one of items 1′-7′, wherein the FGFR inhibitor is selected from erdafitinib, rogoratinib, AZD4547, BGJ398, BLU554, LY2874455, ASP5878, and ARQ087.
9′. The method of any one of items 1′-8′, wherein the FGFR inhibitor is erdafitinib.
10′. The method of any one of items 1′-9′, wherein the cancer spheroid is a spheroid prepared by culturing a cell population obtained from a cancer tissue of the patient in a medium containing a ROCK inhibitor, a TGF-β type I receptor inhibitor, EGF and bFGF.
11′. The method of item 10′, wherein the medium further comprises an agent selected from an adenosine receptor agonist, a PPAR agonist, a calcium-activated potassium-channel (KCa) activator, an NMDA-type glutamate receptor agonist, a histamine H3 receptor agonist, and a retinoic acid receptor (RXR) β2 agonist.
12′. The method of item 11′, wherein the medium comprises an adenosine receptor agonist.
13′. The method of any one of items 10′-12′, wherein the ROCK inhibitor is Y27632 and the TGF-β type I receptor inhibitor is SB431542.
14′. The method of any one of items 10′-13′, wherein the adenosine receptor agonist is NECA.
15′. The method of any one of items 10′-14′, wherein the cancer tissue is a tissue obtained from a primary lesion.
16′. A method of treating colorectal cancer, comprising administering to a colorectal cancer patient in need thereof an effective amount of an FGFR inhibitor.
17′. The method of item 16′, wherein the colorectal cancer patient is a patient selected by the method of any one of items 1′-15′.
18′ The method of item 16′ or 17′, wherein the method further comprises selecting the colorectal cancer patient by the method of any one of items 1′-15′.
19′. The method of any one of items 16′-18′, wherein the FGFR inhibitor is selected from erdafitinib, rogoratinib,
20′. The method of any one of items 16′-19′, wherein the FGFR inhibitor is erdafitinib.
21′. The method of any one of items 16′-20′, wherein the method further comprises administering an EGFR inhibitor.
22′ The method of item 21′, wherein the EGFR inhibitor is cetuximab.
23′. A pharmaceutical composition for treating colorectal cancer comprising an FGFR inhibitor.
24′. The pharmaceutical composition of item 23′, wherein the pharmaceutical composition is administered to a colorectal cancer patient selected by the method of any one of items 1′-15′.
25′. The pharmaceutical composition of item 23′ or 24′, wherein the FGFR inhibitor is selected from erdafitinib, rogoratinib, AZD4547, BGJ398, BLU554, LY2874455, ASP5878, and ARQ087.
26′. The pharmaceutical composition of any one of items 23′-25′, wherein the FGFR inhibitor is erdafitinib.
27′. The pharmaceutical composition of any one of items 23′-26′, wherein the pharmaceutical composition is used in combination with an EGFR inhibitor.
28′ The pharmaceutical composition of item 27′, wherein the EGFR inhibitor is cetuximab.
29′. An FGFR inhibitor for use in treating colorectal cancer.
30′. The FGFR inhibitor of item 29′, wherein the FGFR inhibitor is administered to a colorectal cancer patient selected by the method of any one of items 1′-15′.
31′. The FGFR inhibitor of item 29′ or 30′, wherein the FGFR inhibitor is selected from erdafitinib, rogoratinib, AZD4547, BGJ398, BLU554, LY2874455, ASP5878, and ARQ087.
32′. The FGFR inhibitor of any one of items 29′-31′, wherein the FGFR inhibitor is erdafitinib.
33′. The FGFR inhibitor of any one of items 29′-32′, wherein the FGFR inhibitor is used in combination with an EGFR inhibitor.
34′. The FGFR inhibitor of item 33′, wherein the EGFR inhibitor is cetuximab.
35′. Use of an FGFR inhibitor for the manufacture of a medicament for treating colorectal cancer.
36′. The use of item 35′, wherein the medicament is administered to a colorectal cancer patient selected by the method of any one of items 1′-15′.
37′. The use of item 35′ or 36′, wherein the FGFR inhibitor is selected from erdafitinib, rogoratinib, AZD4547, BGJ398, BLU554, LY2874455, ASP5878, and ARQ087.
38′. The use of any one of items 35′-37′, wherein the FGFR inhibitor is erdafitinib.
39′. The use of any one of items 35′-38′, wherein the FGFR inhibitor is used in combination with an EGFR inhibitor.
40′. The use of item 39′, wherein the EGFR inhibitor is cetuximab.
The present invention is described in further detail in the following examples, which are not in any way intended to limit the scope of the invention.
From each surgical specimen collected from 141 colorectal cancer (CRC) patients, 2 pieces of tumor (0.5-4 cm3 each) and one piece of normal mucosa (˜4 cm2) were isolated, and stored in the ice-cold washing medium [DMEM/F12 with HEPES and 1-glutamine (Nacalai), 100 units/ml penicillin (Nacalai), 0.1 mg/ml streptomycin (Nacalai), and 10% bovine calf serum (SAFC Biosciences)]. Tissues were processed within 24 h after surgery. A piece of the CRC specimen (0.5-1.0 cm3) or a flap of the normal mucosa (1-2 cm2) was separated from the connective tissue, washed with PBS, and minced using scissors in a 60-mm petri dish. The tissue fragments were digested in 2 ml of collagenase solution [the washing medium supplemented with 0.2% collagenase type I (Thermo Fisher) and 50 μg/ml gentamicin (Thermo Fisher)] for 40-60 min at 37° C., and dissociated by pipetting with a 1 ml-pipette with ˜20 min intervals 2-3 times. Then, cells were filtered through a 100-μm cell strainer (Corning) and collected. The cells were then resuspended in Matrigel (Corning), and placed in the center of each well of the 12-well cell-culture plate (˜1×104-5×104 cells/30 μl per well). After polymerization of Matrigel at 37° C., the cells derived from CRC specimens were cultured with cancer medium [Advanced DMEM/F-12 (Thermo Fisher), 100 units/ml penicillin, 0.1 mg/ml streptomycin, 2 mM 1-glutamine (Nacalai), 10 μM Y27632 (R&D), 10 or 1 μM SB431542 (R&D), 5 μg/ml Plasmocin (Invivogen), and 5% FBS (Thermo Fisher)], and the cells derived from normal mucosa were cultured in eL-WRN medium [Advanced DMEM/F-12, 100 units/ml penicillin, 0.1 mg/ml streptomycin, 2 mM 1-glutamine, 50% L-WRL conditioned medium, 10 μM Y27632, 10 or 1 μM SB431542, 50 ng/ml EGF (Peprotech), 5 μg/ml Plasmocin, and 20% FBS]. The L-WRN CM, which is a conditioned medium of L-WRN cells (College of American Pathologists Consensus Statement 1999. Arch Pathol Lab Med. 2000; 124: 979-94.) was prepared according to a previous report (Gene. 1991; 108: 193-9). In some experiments, to the cancer medium was supplemented with 100 ng/ml bFGF (Peprotech), 50 ng/ml EGF, 1 μM NECA (Sigma), and/or B27 supplement (Thermo Fisher). A spheroid line was considered as successfully established when cancer spheroid culture was expanded to one plate (12 wells in a 12-well plate). At the end of the culture, the number of cells was ˜5×104-2×105 cells/well. The cancer spheroids thus obtained were stored in a freezer until use in each experiment. In the following experiments, the frozen cancer spheroid was thawed and passaged 5-20 times, and trypsinized to prepare cell aggregates each containing a few or few dozen of cells. The cell aggregates were then embedded in Matrigel (Corning) for use in experiments. The established spheroid lines were cultured with the minimum supplements.
Preparation of DNA and Histology Specimens from Spheroids
Spheroids in Matrigel were suspended in Cell Recovery Solution (Corning), and collected in 1.5 ml-tubes. Matrigel was digested at 4° C. with rotation for 30-60 min. Spheroids were centrifuged at 200×g for 5 min, and washed with PBS twice at 4° C. DNA was purified using DNeasy Blood & Tissue Kit (Qiagen). For histological analyses, spheroids were passaged once or twice without trypsinization so that larger cell aggregates were formed. The spheroids thus obtained were embedded in iPGell (Genostaff), and fixed with 4% paraformaldehyde in PBS for 16 h at 4° C.
Formalin-fixed paraffin-embedded specimens were sectioned at 4 μm thickness, and stained with H&E. Histological grades of primary cancer and spheroids were determined according to the WHO guideline and the recommendation of the College of American Pathologists.
Hotspot mutations in 50 cancer-related genes were detected by Macrogen using Ion AmpliSeq Cancer Hotspot Panel v2 (Thermo Fisher) according to our order. The list of sequenced genes and mutations was available from manufacturer's website (http://tools.invitrogen com/downloads/cms_106003. csv). The data sets were analyzed using Integrative Genomics Viewer software (Broad Institute).
Array-based CGH analyses was performed with Agilent SurePrint G3 human CGH microarray 1x1M (G4447A, Agilent, Hachioji, Japan) and Genomic DNA ULS Labeling Kit (#5190-0419, Agilent) according to the manufacturer's instructions. Human genomic DNAs (G1521 and G1471, Promega, Madison, Wis., USA) were used as references and controls. The array slides were scanned using Agilent G2565BA microarray scanner (Agilent).
A cDNA fragment encoding a firefly luciferase (luc2, Promega) was inserted into the pCX vector that contained a CAG promoter to construct pCX-Luc2. The expression unit (CAG-luciferase) of pCX-Luc2 was inserted into a lentiviral vector (pCDH, System Biosciences) to construct pCDH-CAG-Luc2. Lentiviral particles were prepared according to a protocol in a previous report (Gene. 1991; 108: 193-9) with minor modifications. Briefly, 2×107 of 293FT cells (Thermo Fisher) were seeded on four 10 cm-dishes, and cultured overnight. Cells were transfected with DNA of 40 μg pCDH-CAG-Luc2 plasmid, 26 μg psPAX2 packaging plasmid, and 14 μg pMD2.G envelope plasmid, with the use of Lipofectamine 2000 (Thermo Fisher). Viral particles were harvested on the second and third days after transfection, and precipitated using PEG-it Virus-Precipitation Solution (System Biosciences). Viral particles were resuspended in 2 ml of the washing medium, aliquoted into 1.5 ml-tubes (50 μl each), and stored at −80° C. Spheroids cultured in one well of a 12-well cell-culture plate were trypsinized, and about half of the cells were collected by centrifugation (200×g) in a 1.5 ml-tube. Cells were resuspended in 50 μl of the virus solution containing 10 μg/ml hexadimethrine bromide and 10 μM Y27632, and incubated for 6 h at 37° C. Then, the cells were collected by centrifugation (200×g), resuspended in 60 μl of Matrigel, and seeded into two wells of a 12-well cell-culture plate.
For growth monitoring assays, luciferase-expressing spheroids were cultured in 2-3 wells of a 12-well cell-culture plate. Spheroids were trypsinized, filtered through a 40-μm cell strainer (Corning), and diluted with the washing medium 4-8 times (based on the volume of Matrigel) to provide a concentration of 5×104-2×106 cells/ml depending on the growth rate and spheroid density. The spheroid cells were resuspended in Matrigel, distributed to the wells of 96-well “white” cell-culture plates (Corning; 3 μl per well) on a plate heater at 37° C., and cultured overnight in 100 μl/well of the medium. Spheroids were rinsed with 100 μl/well of PBS, and incubated with 50 μl of 150 μg/ml luciferin in phenol red-free DMEM/F12 medium (Nacalai) for 10 min at room temperature (20-2° C.). Luminescence was scored using a conventional imaging device (Gel Doc XR+, BioRad). After this initial measurement, spheroids in each well were rinsed with 100 μl of PBS, and cultured in 100 μl of the medium. Luminescence was determined daily or 3 days later. The cell growth rate for each well was estimated as the proportion of photon counts to those on day 1. Four or six replicates (with similar statistical power) were analyzed for each data point in all experiments except for the chemical compound screening assays.
To culture patient-derived tumor initiating cell spheroids of colorectal cancer (PD-CRC-TIC spheroids), we prepared the “cancer medium” in which the basal medium (Advanced DMEM/F12) was supplemented with FBS (5%) and a Rock inhibitor Y27632 and a TGF-β type I receptor inhibitor SB431542. To culture spheroids of normal colonic epithelial cells from the same patients, we also prepared “the eL-WRN medium” by supplementing the L-WRN conditioned medium with Y27632 and SB431542. We cultured 148 colorectal cancer samples from 141 colorectal cancer patients. Pathologically, the samples were classified as low-grade (138/148), high-grade (5/148), and mucinous (5/148) adenocarcinomas according to the standard histological criteria. The histological features of the cancer epithelium coincided well between the primary tumors and their spheroids (
It was difficult to determine the number of live cells in 3D culture and the growth rate of the cells. To determine the number of cells in spheroid culture precisely, we utilized spheroids infected with a lentivirus containing a luciferase expression vector driven by CAG promoter. Because high efficiency of infection eliminated the necessity to enrich expressor subpopulations, we passaged post-infection spheroids 2-3 times without subcloning, and directly subjected them to cell growth assays. We monitored the growth of the normal and cancer spheroid lines daily from post-passage days 1 to 4 (
Stimulation of CRC-TIC Spheroid Growth with EGF and bFGF
To investigate whether EGF and bFGF helped spheroid proliferation in the cancer medium as described above, we cultured with EGF and/or bFGF four slow-growing CRC-TIC spheroid lines that carried wild-type RAS/RAF genes (HC6T, HC9T, HC11T, HC21T). Because EGF/bFGF facilitated the growths of these spheroid lines substantially (
To find additional supplementary factors that could promote spheroid growths, we screened 80 pharmacologic agonists or activators for growth-promoting effects on three luciferase-expressing CRC-TIC spheroid lines with moderate growth rates (i.e., 3-4 times in 3 days) (HC21T, HC18T, HC25T). We identified eleven compounds that stimulated spheroid growths by >25% (
We then performed titration experiments for GW0742 (PPARδ agonist), NECA (non-selective AR agonist), and SKA-(activator of KCa2 and KCa3.1a). While NECA exhibited a strong activity of growth promotion (EC50=63.1 and 195 nM in HC18T and HC21T spheroid lines, respectively), other two compounds showed significant effects at 10 μM (
Stimulation of CRC-TIC Spheroid Growth with NECA Through cAMP-PKA Signaling Pathway
Because A2B is the only the adenosine receptor expressed in the colonic epithelium, NECA appears to stimulate the cyclic AMP (cAMP)-protein kinase A (PKA) signaling in these cells. Consistently, NECA as well as CV1808 induced morphological changes in spheroids characterized by cell flattening and increased sphere size, similar to those in forskolin-treated spheroids (
Supplementation with NECA in addition to EGF and bFGF enabled us to stably maintain three slow-growing spheroid lines (HC28T, HC32T, HC52T), for which EGF and bFGF alone were ineffective in growth stimulation. Accordingly, we infected these spheroid lines with a lentiviruses and make them express a luciferase gene to monitor their growth. By the luciferase assay, we confirmed that addition of EGF, bFGF and NECA significantly stimulated their growths by 50% or more (
On the basis of the above results, we modified culture media for both normal and cancer SC spheroids. In the periods A-D in Table 1, EGF and bFGF were added to the standard medium only when the growth with the medium was insufficient. We optimized its concentration of SB431542 in the period V to 1 μM to avoid its growth inhibition often seen at 10 μM (
The overall success rate for establishing CRC-TIC spheroids was 73% (108/148). The reasons for unsuccessful CRC-TIC spheroid culture included contamination by microorganisms (bacteria or yeasts; 4 cases), cell damage by radiation therapy before surgery (2 cases), and poor cell growth followed by cell death in early passages (of unknown causes; 34 cases). We found no statistical correlations between the success rates and tumor stages (Table 2). As a result of the optimizations of culture conditions, the success rate for establishing CRC-TIC spheroids increased to ˜90% in the period E in Table 1 (Table 1).
To compare PDX model and PDSX model in application with drug sensitivity tests, we established PDX and PDSX models from 92 samples obtained from 89 colorectal cancer patients who underwent surgical resections.
Tumor samples were washed with the washing medium (DMEM/F12 containing 10% bovine calf serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin) and PBS twice each. Necrotic tissues of the tumor samples were removed as much as possible. After washing, we divided the tumor samples into cubic fragments of 50-100 mm3 and used to establish cancer spheroids and PDXs.
To establish P0 (founder) PDXs, the tumor fragments were implanted directly to each flank of nude mice and/or NOD-SCID mice with under isoflurane anesthesia. The growth of implanted tumors was measured with a pair of calipers at least once a week, and the volume of each tumor was estimated using the following formula; tumor volume (mm3)=[length (mm)×width2 (mm2)]/2 (the length means the longer tumor axis and the width means the shorter tumor axis). Implantations were judged as successful when estimated tumor volumes reached 1000 mm3, and as failed if no visible tumor masses were recognized for 6 months after transplantation. To passage the tumors, we excised them and divided them into cubic fragments of 50-100 mm3. These fragments were transplanted into nude mice to establish the next generation of PDXs.
Patient-derived spheroids used in this example were obtained by thawing the frozen samples of the 21 lines established in Example 1 using the cancer medium, which is a basic medium (Advanced DMEM/F-12) supplemented with FBS (5%), a ROCK inhibitor (Y27632) and a TGF-β type I receptor inhibitor (SB431542), or the cancer medium further comprising EGF and bFGF. The spheroids were cultured with the medium used when they were established.
PD-CRC-TIC spheroids were cultured in a 12-well culture plate. Spheroids in confluent culture (4×105-4×106 cells/well) were rinsed with PBS twice, and transferred to a 1.5-ml tube together with Matrigel. The volume of the spheroid suspension was adjusted to 100 μl with PBS, and the suspension thus obtained was injected into nude mice subcutaneously at 1×105-9×105 cells/mouse.
We successfully established P0 (founder) generation PDX mice in 56 of 92 cases (61%), and PD-CRC-TIC spheroids in 68 of 92 cases (74%).
To determine the efficiency in establishing xenograft models from the PD-CRC-TIC spheroids, we injected nude mice with 21 PD-CRC-TIC spheroid lines subcutaneously, and successfully established model mice with 18 lines (86%). In contrast, the success rate for passaging from the P0 (founder) to P1 generation PDXs was 70%. For drug-dosing tests, a large number of mice enough for a strong statistical power (e.g., n=3-6 for each group) has to be prepared. Accordingly, for PDX models, serial transplantations are inevitable with two or more passages. On the other hand, for PDSX models, we can generate enough numbers of PDSX mice simultaneously once we propagate spheroids in vitro.
In 56 cases that generated P0 (founder) PDXs, 148 of 236 (63%) samples formed P1 PDXs. In contrast, 198/228 (87%) of spheroid-injected mice developed PDSX mice. PDSX models showed much smaller variances in tumor volumes among individual mice than PDX models. The coefficients of variation (CVs) for P1 PDXs and PDSXs were 0.53 and 0.29, respectively.
To evaluate the reliability in drug sensitivity tests with PDX and PDSX models, we prepared test mice transplanted with samples from 4 colorectal cancer patients. We had to exclude 23 of 94 (24%) PDXs as outliers because of the large individual variances in growth rates. In contrast, we eliminated 9 of 85 (11%) PDSXs according to the same criteria.
First, we prepared PDX and PDSX models derived from a CRC case (HC13T) that carried a BRAFV600E mutation, and performed a drug sensitivity test (
PDX (P2-P4) and PDSX mice were prepared and subjected to the drug sensitivity test when the estimated tumor volumes reached 300-500 mm3. Mice were distributed into the control and treatment groups (n=3-6 per group). The day of the dosing start was defined as day 1, and the tumor volume and the body weight of each mouse were measured twice a week for 3 weeks (day 1-22). The treatment protocols were designed to reflect clinical regimens and drug doses for mice were calculated according to the following formula: mouse dose (mg/kg)=human dose (mg/kg)×37 (human Km)/3 (mouse Km). For the FOLFOX-like treatment, mice were injected intraperitoneally (i.p.) with oxaliplatin (12 mg/kg) and levofolinate calcium (30 mg/kg) first, followed by 5-fluorouracil (55 mg/kg), weekly for 3 weeks. For cetuximab treatment, mice were injected intraperitoneally (i.p.) at 250 μg per injection, twice a week for 3 weeks. These doses were less than the maximal tolerated doses and approximately 80% of the clinically relevant doses. Relative tumor volumes were calibrated to the initial tumor volumes on day 1. To evaluate effects of treated drugs, T/C (Treated/Control) and TGI (Tumor Growth Inhibition) values were calculated. The T/C was defined as the ratio of relative tumor volume in a treated group to that in the untreated group, which indicates tumor growth inhibition by a treatment. The value of T/C was calculated by the following formula. The value of TGI was also calculated by the following formula and used to assess tumor growth inhibition.
T/C (%)=relative tumor volume (treated)/relative tumor volume (control)×100.
TGI (%)=[1−Δrelative tumor volume (control)/Δrelative tumor volume (treated)]×100,
or
TGI (%)=[1−Δrelative tumor volume (control)]×100 for Δrelative tumor volume (control)<0 (tumor regression).
Δrelative tumor volume=(relative tumor volume on day 22)−(relative tumor volume on day 1).
Cetuximab was ineffective in both PDX and PDSX models (T/C; 120% and 114% with PDXs and PDSXs, respectively), the FOLFOX-like regimen significantly decreased the tumor growth (T/C; 48% and 57% with PDXs and PDSXs, respectively) (
To investigate the reproducibility, we performed drug sensitivity tests with two sets of PDSXs (spheroids at 12th and 16th passages; P12 and P16) and PDXs (P2 and P3 generations) independently prepared from HC13T tumor. The relative tumor volumes of the two sets with PDSXs showed a stronger correlation [Spearman r=0.99, 95% CI: 0.96-0.99] than those with PDXs [Spearman r=0.87 95% CI: 0.69-0.95] (
(2) Drug Sensitivity in PDSX Models that Reflects Clinical Outcome of Patients
We compared drug responses of PDSXs with the clinical outcome of the patients in a retrospective manner. We prepared panels of PDSXs from seven patients who had been treated with chemotherapies against metastases after resection of their primary tumors. We performed a total of nine drug-dosing tests that matched chemotherapeutic regimens in the corresponding patients. Notably, eight of the nine sets (89%) of the drug-dosing results with PDSXs well reflected their corresponding clinical outcomes. Namely, the therapeutic regimens that were efficacious in the patient [partial response (PR) or stable disease (SD) with RECIST] were also effective in treating PDSXs, whereas those that were not efficacious in the patients [progressive disease (PD) with RECIST] were ineffective in PDSXs. In addition, there was a strong correlation between the results of PDSX dosing tests (Treated/Control, T/C) and patient outcomes [the best response with RECIST (BR) (r=0.70, p=0.04) and the durations of clinical responses in patients (DOR) (r=−0.77, p=0.04)]. A strong correlation was also observed between TGI (Tumor Growth Inhibition) and patient outcomes [both BR (r=−0.72, p=0.03) and DOR (r=0.83, p=0.02)].
Results of representative patients are shown in
Patient 1 in
Patient 3 in
The PDX models have been reported to be predictive of clinical responses. To further evaluate the predictive power of PDSXs, we compared the results of drug dosing tests (T/C and TGI) on PDSXs with those on PDXs derived from the same patient tumors. The results of PDSXs showed a strong concordance with those of PDXs in both T/C [r=0.93 (95% CI: 0.63 to 0.99), p=0.001] and TGI [r=0.93 (95% CI: 0.64 to 0.99), p<0.001]. These results support the robust reliability of PDSXs in non-clinical evaluation of drug sensitivity.
One of the most practical limitations of PDXs for personalized medicine is the long time that is needed to prepare enough numbers of PDX mice for drug-dosing tests. For example, it took 5 months or longer to prepare P2 generation PDXs in our study. [More precisely, it took 70 days (median) for the P0 (founder) generation tumors to expand to 1,000 mm3, and ˜50 days for the P1 generation to reach the same size]. Thereafter, it took us a month for P2 PDXs to grow up to the size of 300-500 mm3 (appropriate for drug dosing). In contrast, it took us for only 2-3 months to prepare a group of ˜20 PDSXs mice. [˜a month to establish spheroids from the primary lesion followed by 1-2 more weeks to expand them to enough numbers of spheroids for injection into mice.] Thereafter, it took ˜24 days to expand PDSXs to the size of 300-500 mm3. These results indicate that the PDSX method is far more advantageous than the PDX method not only in the accuracy of drug sensitivity testing but also in saving time for preparation of the tumor-bearing mice. We can expect to feedback the dosing data to the bedside before deterioration of the patient conditions.
We established 111 cancer cell spheroid lines and paired 11 normal epithelial cell spheroid lines from surgical specimens of 110 colorectal cancer patients. One of the patients had double cancers.
The 94 cancer spheroids established in the examples with the cancer medium (a basic medium (Advanced DMEM/F12) supplemented with FBS (5%), a ROCK inhibitor (Y27632) and a TGF-β type I receptor inhibitor (SB431542)) with or without EGF and bFGF were cultured in the same medium as that used at the time of establishment. The spheroids of normal colon epithelial cells from the same patients were cultured in “eL-WRN medium”, in which Y27632 and SB431542 were added to L-WRN conditioned medium. DNA was extracted from about 2×105 to about 1×106 cells from each spheroid.
For all the 111 patients, we employed an on-chip MSI test that analyzed PCR products amplified from DNA extracted from cancer and normal spheroids on Agilent Bioanalyzer chips. The MSI status was determined using five microsatellite markers BAT25, BAT26, D5S346, D2S123, and D17S250 (Bethesda panel). In microsatellite stable (MSS) cases, the peak patterns of PCR products were concordant closely between cancer cells and paired normal cells (
Next, to confirm the correlation between MSI status and MMR deficiency, immunohistochemistry (IHC) of MMR proteins (MLH1, MSH2, MSH6 and PMS2) were performed in primary lesion of all 111 cases. As a result, 8 MMR deficiency cases and 87 MMR proficient cases were detected (
Next, we employed sequence analysis to determine mutations in mononucleotide repeats in TGFBR2, BAX, IGF2R and CAPSPS. The high purity of the spheroid-derived DNA samples allowed us to detect heterozygous mutations (
To compare the quality of spheroid-derived DNA samples to that of formalin-fixed and paraffin-embedded (FFPE) tissue-derived DNA samples, we performed the on-chip MSI test with FFPE tissue-derived DNA samples and spheroid-derived DNA samples in 52 cases. First, the quality of both DNA samples was checked. The median A260/A280 of spheroid-derived DNA samples was significantly higher than that of FFPE tissue-derived DNA samples (1.8 vs 1.7, p<0.0001), and the median concentration of spheroid-derived DNA samples was also higher than that of FFPE tissue-derived samples (44 ng/μl vs 30 ng/μl, p=0.06) (
For all loci tested, the peak heights of PCR products amplified from the spheroid-derived DNA were significantly higher than those of FFPE tissue-derived DNA (
To test the quality of the DNA samples further, we performed sequencing of MMR deficiency target 4 genes using spheroid derived-DNA samples and FFPE tissue-derived DNA samples in MSI-H cases. The heterozygous mutations detected with spheroid derived-DNA samples were not confirmed with FFPE tissue-derived DNA samples (
Erdafitinib (JNJ42756493, Active Biochem, pan-FGFR inhibitor), AZD4547 (Active Biochem, FGFR1-3 inhibitor), BGJ398 (AdooQ, FGFR1-3 inhibitor), BLU-554 (Selleck Chemicals, FGFR4 selective inhibitor), and rogoratinib (MedChemo Express, FGFR1-3 inhibitor) were dissolved and diluted in DMSO. Cetuximab (Erbitux, Merck) was dissolved in phosphate buffered saline (PBS).
Spheroids used in this example were the patient-derived cancer spheroids established by culturing tumor-derived cells in the cancer medium containing 100 ng/ml bFGF (Peprotech) and 50 ng/ml EGF (Peprotech) and the spheroids established from normal mucosa cells in Example 1. The established cancer spheroid lines were cultured with the aforementioned cancer medium containing bFGF and EGF.
Luciferase-expressing spheroids were prepared as described in Example 1 by using a lentiviral vector.
Luciferase-expressing spheroids were cultured in 2-3 wells of a 12-well cell-culture plate (in 60-90 μl Matrigel). Spheroids were trypsinized, filtered through a 40-μm cell strainer (Corning), and diluted with the washing medium 4-8 times (based on the volume of Matrigel) to provide a concentration of 5×104-2×106 cells/ml depending on the growth rate and spheroid density. From the spheroid solution thus obtained, 300 μl was taken and the cells were resuspended in an equal volume of Matrigel. The spheroid cells in Matrigel were distributed to the wells of 96-well “white” cell-culture plates (Corning; 3 μl per well) on a plate heater at 37° C., and cultured overnight in 100 μl/well of the medium. Spheroids were rinsed with 100 μl/well of PBS, and incubated with 50 μl of 150 μg/ml luciferin in phenol red-free DMEM/F12 medium (Nacalai) for 10 min at room temperature (20-28° C.). Luminescence was scored using a conventional imaging device (Gel Doc XR+, BioRad). After this initial measurement, spheroids in each well were rinsed with 100 μl of PBS, and cultured in 100 μl of the medium. Luminescence was determined on day 1 and day 3. Effects of a drug was determined by obtaining GEI (growth effect index), which is the growth rate of a group treated with the drug compared to a control group (a solvent-treated group). Results were shown using means in three independent experiments each conducted in triplicate.
Patient-derived spheroids were cultured in a 12-well culture plate to become confluent (4×105-4×106 cells/plate) and the spheroids were rinsed twice with PBS and placed in a 1.5 ml tube with Matrigel. The volume of the spheroid suspension was adjusted to 100 μl with PBS and the suspension thus obtained was subcutaneously injected into 4-6 week female nude mice (BALB/c-nu) at 1×105-9×105 cells/mouse.
PDSX mice were subjected to drug-dosing test when the mean estimated tumor volume reached 300 mm3. Tumor volume (mm3) was estimated by the following formula: length×width2/2. We excluded the following kinds of tumors from the tests as outliers; too small (<100 mm3), spontaneously shrinking, remaining unchanged in size, or deeply implanted and difficult to be measured.
Mice in each set were distributed into the control and treatment groups (n=3-4 per group), with the latter included the followings; erdafitinib monotherapy, cetuximab monotherapy, and erdafitinb+cetuximab. The day of the dosing start was defined as day 0, with the tumor volume of each mouse measured twice a week for 3 weeks (days 0-21). For erdafitinib treatment, the mouse dosage was set as 10 mg/kg according to the previous report (Perera T P S et al. Mol Cancer Ther. 2017; 16(6): 1010-20), and mice were dosed orally by allowing them to take a gel (MediGel® Sucralose, ClearH2O) containing the drug at 10 mg/mL in DMSO ad libitum as water substitute. For cetuximab treatment, the mouse dosage was set according to the following formula: mouse dose (mg/kg)=human dose (mg/kg)×37 (hKm)/3 (mKm) where Km indicates human or mouse body surface coefficient, and mice were injected intraperitoneally (i.p.) at 250 μg per injection, twice a week for three weeks.
The relative tumor volume was estimated by calibration to the initial tumor volume on day 0. To evaluate the drug dosing effects, the ratio of tumor volume on day 21 to that on day 0 was compared among the four groups.
Paraffin-embedded tissues of PDSXs were prepared according to the standard procedures. Sections were stained with H&E or immunostained for Ki67.
Preparation of DNA from Spheroids
Spheroids in Matrigel were suspended in Cell Recovery Solution (Corning), and collected in 1.5 ml-tubes. Matrigel was digested at 4° C. with rotation for 30-60 min. Spheroids were centrifuged at 200×g for 5 min, and washed with PBS twice at 4° C. DNA was purified using DNeasy Blood & Tissue Kit (Qiagen).
Array-based CGH analysis was performed with Agilent SurePrint G3 human CGH microarray 1x1M (G4447A, Agilent, Santa Clara, Calif., USA) and Genomic DNA ULS Labeling Kit (#5190-0419, Agilent) according to the manufacturer's instructions. Human genomic DNAs (G1521 and G1471, Promega, Madison, Wis., USA) were used as references and controls. The array slides were scanned using Agilent G4900DABA microarray scanner (Agilent).
Preparation of RNA from Spheroids
Spheroids in Matrigel were suspended in Cell Lysis Solution (TaKaRa), and RNA was purified using NucleoSpin® RNA kit (#U0955C, TaKaRa).
Array-based mRNA expression analysis was performed with SurePrint G3 Human Gene Expression 8×60k version 3.0 (G4851C, Agilent) and Quick Amp Labeling Kit (#5190-0442, Agilent) according to the manufacturer's instructions. The array slides were scanned using Agilent G4900DA SureScan microarray scanner (Agilent).
Data were analyzed using one-way and two-way ANOVA followed by Tukey's post-test using GrafPad Prism ver.6 (GraphPad software. Inc.).
The spheroid lines shown in Table 3 were used in this example. We first treated three spheroid lines (HC6T, HC9T, and HC20T) with an FGFR inhibitor (erdafitinib, AZD4547, BGJ398, BLU554 or rogoratinib), and determined GEI. These spheroid lines (HC6T, HC9T and HC20T) contained no hot-spot mutations in any of KRAS, NRAS and BRAF genes (Table 3). Also, the growth of HC6T and HC9T was heavily dependent on bFGF (data not shown). As shown in
Amino acid replacements are shown with amino acids before and after replacement at the affected codon.
Note that 50.5% of mutations in the APC gene was detectable in the test.
Well, well differentiated adenocarcinoma; Mod, moderately differentiated adenocarcinoma; Poor, poorly differentiated adenocarcinoma: Muc, mucinous carcinoma.
We then tested six spheroid lines that did not depend on bFGF for in vitro growth (HC1T, HC10T, HC11T, HC73T, HC5T and HC13T). Erdafitinib showed no growth inhibition on the six spheroid lines at clinically relevant doses (0.01-1 μM) (
We tested effects of a combination of FGFR and EGFR inhibitors on the colorectal cancer spheroid growth. Among EGFR inhibitors, anti-EGFR antibodies cetuximab and panitumumab have already been approved for the treatment of RAS/RAF mutation-free colorectal cancer. However, cetuximab failed to suppress the in vitro growth of spheroids (
Erlotinib showed dose-dependent growth inhibition on RAS/RAF mutation-free spheroid lines (HC6T, HC9T, HC11T, and HC20T) (
Furthermore, we screened for the effects of the combination of erdafitinib and erlotinib on other seven spheroid lines that were free of RAS/RAF hot-spot mutations including the four that were almost insensitive to erdafitinb alone (HC1T, HC10T, HC11T, and HC73T;
(3) Reduction of PDSX Tumor Sizes with Erdafitinib and/or Cetuximab at Clinically Relevant Doses
To determine the potential efficacy in blocking the FGFR and/or EGFR signaling in tumor-bearing mice, we established PDSX mice from two colorectal cancer spheroids lines, HC6T and HC20T, that responded well to both FGFR and
EGFR inhibitors in vitro (
In HC6T-PDSX mice, cetuximab monotherapy, erdafitinib monotherapy and erdafitinib+cetuximab combination reduced the tumor volume with statistically significant differences from the control tumors (p<0.05-0.0001;
(4) FGF and FGFR Gene Copy Numbers and mRNA Expression Levels in Patient-Derived Colorectal Cancer Spheroids
We examined whether there are changes in the copy numbers of the FGF9 and FGFR1-4 genes in patient-derived colorectal cancer spheroids. As shown in Table 4, no increase in the copy numbers of FGFR1 to 4 (normal value was 2) was observed in HC6T, HC9T, and HC20T, in which the FGFR inhibitor was effective alone. An increase in copy number was observed with HC11T, in which the FGFR inhibitor had no effect. As for FGF9, an increase in copy number was observed in HC6T and HC9T, in which the FGFR inhibitor was effective, and in HC11T and HC73T, in which the FGFR inhibitor was not effective. Further, mRNA expression levels of FGF3, 9, 19 and 20 and FGFR1 to 4 were examined, but no correlation with the response to the FGFR inhibitor was observed (
| Number | Date | Country | Kind |
|---|---|---|---|
| 2017-236374 | Dec 2017 | JP | national |
| 2018-195647 | Oct 2018 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2018/044894 | 12/6/2018 | WO | 00 |
