A method for drug sensitivity detection of primary tumor organoids
By combining light microscopy and fluorescence information detection methods, the accuracy problem of drug sensitivity testing in tumor organoids has been solved, enabling rapid and accurate drug sensitivity assessment, which is applicable to drug sensitivity testing of primary tumor organoids in various cancer types.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANGHAI ONETAR BIOMEDICINE CO LTD
- Filing Date
- 2022-12-27
- Publication Date
- 2026-06-16
AI Technical Summary
In the existing technology, single tumor organoid drug sensitivity detection methods cannot accurately reflect the sensitivity of different tumor organoids to drugs, and the culture of primary tumor organoids is complex, with interference from impurities such as fibroblasts and immune cells, resulting in poor timeliness.
A detection method combining light microscopy and fluorescence information was adopted. By acquiring and calculating parameters such as the long diameter, area, light transmittance, smoothness of the outer curved surface, morphological integrity, and fluorescence staining intensity of tumor organoids, drug sensitivity was detected using a biochip to comprehensively evaluate the growth and viability of each organoid.
It provides a reliable and rapid method for drug sensitivity testing, which can provide patients with timely medication references, reduces interference from impurities, is applicable to a variety of cancers, and has high accuracy in test results, making it suitable for drug development and clinical use.
Smart Images
Figure CN115876739B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of organoids, and more particularly to a method for detecting the drug sensitivity of primary tumor organoids. Background Technology
[0002] Tumor metastasis and drug resistance are leading causes of death in cancer patients. To achieve precision medicine with more effective treatments, preclinical models representing tumor characteristics are needed to provide a research foundation. Human tumor organoids, as models for in vitro drug sensitivity testing, have opened up new avenues for this purpose. Due to tumor heterogeneity, different patients may respond differently to the same antitumor drugs. Using organoid models for drug sensitivity testing can allow for personalized selection of the optimal treatment regimen, maximizing therapeutic efficacy and minimizing adverse reactions.
[0003] Current tumor organoid drug sensitivity testing mainly uses passaged organoids, while primary tissue takes a long time from organoid formation to passage. This results in poor timeliness for cancer patients, especially those with metastases, and cannot meet their clinical needs.
[0004] On the other hand, current methods for interpreting drug sensitivity results of tumor organoids mainly employ single image analysis or chemical colorimetric methods. However, the culture conditions of primary tumor organoids are complex, with interference from fibroblasts, immune cells, necrotic cells, and a large number of impurities that can affect the interpretation of drug sensitivity results. Therefore, using a single detection method cannot accurately reflect the sensitivity of different tumor organoids to drugs. Summary of the Invention
[0005] The main objective of this invention is to solve the problem that existing single detection methods cannot accurately reflect the sensitivity of different tumor organoids to drugs.
[0006] The first aspect of the present invention provides a method for detecting drug sensitivity, the method comprising:
[0007] Obtain the major diameter L0 of each organ in well D0. n Area S0 n Observation of transmittance T0 n Surface smoothness R0 n Morphological integrity C0 n ;
[0008] Obtain the major diameter L4 of each organ in well D4. n Area S4 n Observation of transmittance T4 n Surface smoothness R4 n Morphological integrity C4 nAnd the average fluorescence intensity Fg within the area of organoids determined by live and dead fluorescence staining. n and Fr n ;
[0009] Calculate the normalized score change values for each organoid corresponding to wells D0 and D4 using the following formula:
[0010]
[0011] in,
[0012] n represents the number of organoids ranging from 30µm to 150µm in each pore;
[0013] P and P control These represent the sum of organoid scores for the drug administration well and the control well, respectively.
[0014] L4 n D4 represents the major diameter of each organ in each well, and A4 represents the major diameter of each organ in each well. n Viability of various organs in each well of D4 as shown by fluorescent staining;
[0015] L0 n Let D0 be the major diameter of each organ in each orifice, and A0 be the minor diameter of each organ in each orifice. n The viability of each organ in each well of D0 was calculated based on morphological parameters;
[0016] A0 n The viability of each organ in each well of D0 was calculated based on morphological parameters;
[0017] A4 n The viability of each organ in well D4 as indicated by fluorescent staining was calculated using the following formula:
[0018] A4 n =Fg n / (Fg n +Fr n )
[0019] A0 n The following was calculated by fitting a function to morphological parameters and fluorescence display activity:
[0020] A0 n =c(M0) n )
[0021] Among them, M0 n The calculation formula is as follows:
[0022]
[0023] Among them, T0 n Transmittance and R0 were observed for each organ in D0 well. n For D0, the smoothness of the outer curved surface of each organ in each well, C0n D0 represents the morphological integrity of various organs in each well; Tb, Rb, and Cb are the baseline observed transmittance, baseline outer surface smoothness, and baseline organ morphological integrity, respectively, calibrated by good organoids observed under the same optical parameters.
[0024] The formula for calculating c is as follows:
[0025]
[0026] in,
[0027]
[0028] Among them, T4 n Transmittance of various organs was observed in each well of D4, and in R4... n For the smoothness of the outer curved surface of each organ in D4, C4 n The integrity of the morphology of various organs in each well of D4.
[0029] A biochip that performs the drug sensitivity detection method for primary tumor organoids.
[0030] This invention can be widely applied to the detection and interpretation of drug sensitivity in organoids of primary tumors of different cancer types. It is not only suitable for drug development, but also provides timely and reliable evidence for clinical drug use.
[0031] This invention overcomes the problems of limited dimensions and low confidence in previous organoid drug sensitivity interpretation methods. By combining light microscopy information with fluorescence information, it comprehensively provides important information such as the growth status and viability of each organoid in the field of view, making it a reliable new method for detecting organoid drug sensitivity.
[0032] The detection method described in this invention is highly versatile and has a wide coverage. It provides targeted measurement methods for most types of cancer and can minimize the interference of fibroblasts, immune cells, and other contaminating cells on the interpretation of quantitative drug sensitivity results.
[0033] This invention offers rapid and convenient testing. With a well-established organoid culture system, drug sensitivity testing of primary cultured organoids can be completed in about two weeks, providing timely medication references for patients requiring postoperative adjuvant or neoadjuvant therapy. Attached Figure Description
[0034] Figure 1 A morphological diagram of an organoid in good condition;
[0035] Figure 2 The image shows the morphology of organoids with low light transmittance.
[0036] Figure 3 A diagram showing the morphology of organoids with rough outer curved surfaces:
[0037] Figure 4 The image shows the morphological features of organoids with poor morphological integrity.
[0038] Figure 5 The experimental results of the control group of squamous cell carcinoma of the tongue root are shown in the figure.
[0039] Figure 6 Figure showing the experimental results of everolimus treatment in the squamous cell carcinoma organoid group at the base of the tongue;
[0040] Figure 7 This is a graph showing the tumor inhibition rate of organoids for squamous cell carcinoma of the tongue base.
[0041] Figure 8 Figure showing the experimental results of organoids for nasopharyngeal malignant tumors (control group).
[0042] Figure 9 Figure 1 shows the experimental results of the gemcitabine + docetaxel group of organoids for nasopharyngeal malignant tumors.
[0043] Figure 10 Figure 1 shows the experimental results of the vincristine + actinomycin + cyclophosphamide drug group for organoids of malignant tumors in the nasopharynx.
[0044] Figure 11 This is a graph showing the tumor inhibition rate of organoids for malignant tumors in the nasopharynx.
[0045] Figure 12 Figure showing the experimental results of the control group of colorectal cancer organoids;
[0046] Figure 13 Figure showing the experimental results of the group treated with regorafenib plus sintilimab in colorectal cancer organoids;
[0047] Figure 14 Figure showing the experimental results of the TAS102 drug-treated colorectal cancer organoid group;
[0048] Figure 15 This is a graph showing the tumor inhibition rate of organoids for colorectal cancer.
[0049] Figure 16 Figure showing the experimental results of organoids for oropharyngeal malignant tumors (control group).
[0050] Figure 17 Figure showing the experimental results of everolimus treatment in oropharyngeal malignant tumor organoids;
[0051] Figure 18 Figure 1 shows the experimental results of the group treated with palbociclib plus apeliximab in organoids for oropharyngeal malignant tumors.
[0052] Figure 19 This is a graph showing the tumor inhibition rate of organoids for oropharyngeal malignant tumors. Detailed Implementation
[0053] This invention provides a method for detecting the drug sensitivity of primary tumor organoids. The method includes: obtaining the major diameter L0 of each organ type in well D0. n Area S0 n Observation of transmittance T0 n Surface smoothness R0 n Morphological integrity C0 n Obtain the major diameter L4 of each organ in well D4. n Area S4 n Observation of transmittance T4 n Surface smoothness R4 n Morphological integrity C4 n And the average fluorescence intensity Fg within the area of organoids determined by live and dead fluorescence staining. n and Fr n The normalized score change values of each organoid corresponding to wells D0 and D4 are calculated according to the formula. This invention overcomes the problems of limited dimensions and low confidence in previous organoid drug sensitivity interpretation methods. It combines light microscopy information with fluorescence information to comprehensively provide important information such as the growth status and viability of each organoid in the field of view. It is a reliable new method for detecting organoid drug sensitivity.
[0054] The terms “first,” “second,” “third,” “fourth,” etc. (if present) in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms “comprising” or “having,” and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.
[0055] The drug sensitivity detection method for primary tumor organoids described in this invention can be used for various types of tumor organoids. The tumor tissue of the tumor organoids is selected from any one of the following: colon cancer, rectal cancer, gastric cancer, esophageal cancer, oral cancer, osteosarcoma, prostate cancer, breast cancer, brain cancer, neuroendocrine tumors, liver cancer, kidney cancer, pancreatic cancer, lung cancer, melanoma, multiple myeloma, ovarian cancer, cervical cancer, skin cancer, duodenal cancer, gallbladder cancer, intrahepatic and extrahepatic bile duct cancer, glioma, head and neck squamous cell carcinoma, synovial sarcoma, liposarcoma, fibrosarcoma, pleomorphic undifferentiated sarcoma, laryngeal papilloma, nasopharyngeal carcinoma, pancreatic neuroendocrine tumor, endometrial cancer, hemangioendothelioma, bladder cancer, ureteral tumor, adrenal tumor, and thyroid cancer tissue.
[0056] A method for detecting drug sensitivity in primary tumor organoids, characterized in that the method comprises:
[0057] Obtain the major diameter L0 of each organ in well D0. n Area S0 n Observation of transmittance T0 n Surface smoothness R0 n Morphological integrity C0 n ;
[0058] Obtain the major diameter L4 of each organ in well D4. n Area S4 n Observation of transmittance T4 n Surface smoothness R4 n Morphological integrity C4 n And the average fluorescence intensity Fg within the area of organoids determined by live and dead fluorescence staining. n and Fr n ;
[0059] Calculate the normalized score change values for each organoid corresponding to wells D0 and D4 using the following formula:
[0060]
[0061] Drug sensitivity testing also includes the primary tumor organoid culture step:
[0062] Wash tumor tissue specimens with pre-cooled cleaning solution;
[0063] Add digestive fluid and incubate for 30-120 minutes;
[0064] Add serum-containing PBS to stop digestion, centrifuge, and discard the supernatant.
[0065] Add DNaseI; centrifuge, resuspend cells and filter using a filter membrane, discard the supernatant after centrifugation;
[0066] The cell pellet was then treated with erythrocyte lysis buffer.
[0067] Transfer the resuspended cell suspension into EP tubes;
[0068] Wash with pre-cooled cleaning solution, centrifuge, add an appropriate amount of matrix gel and mix well, then dispense and plate the material, add organoid culture medium and culture to obtain primary tumor organoids.
[0069] like Figure 1-4 , Figure 1 A morphological diagram of an organoid in good condition. Figure 2 For organoid morphology images with low light transmittance, Figure 3 The morphological diagram of organoids with rough outer curved surfaces. Figure 4The images show the morphological characteristics of organoids with poor morphological integrity. It can be seen that the fluorescent staining method can effectively characterize the morphology of organoids.
[0070] Example 1
[0071] A method for interpreting the drug sensitivity of organoids from squamous cell carcinoma of the tongue base includes the following steps:
[0072] Step 1, Primary squamous cell carcinoma organoid culture of the tongue base: Wash tumor tissue specimens with pre-cooled washing solution.
[0073] Take a photo and cut it to 1mm. 3 Cells were placed in preheated digestion solution and incubated at 37°C with shaking for 30 mins to 2 hours, manually shaking and inverting every 15 minutes to mix. Digestion was stopped by adding serum-containing PBS, followed by centrifugation and discarding the supernatant. DNase I was added and incubated at 37°C for 4 min. After centrifugation, the cells were resuspended and filtered through a 100 μm filter. The supernatant was discarded after centrifugation, and the cell pellet was treated with erythrocyte lysis buffer. The resuspended cell suspension was transferred to a 1.5 ml EP tube. The cells were washed twice with pre-cooled washing buffer and centrifuged at 1500 rpm for 5 min. An appropriate volume of matrix gel was added based on the amount of cell pellet for dispensing and plating.
[0074] Step 2, determine the drug regimen and concentration: The drug concentration used in the drug sensitivity test can be determined by consulting the FDA drug instructions or searching for relevant literature with high impact factors in NCBI to find the Cmax concentration of the drug in the literature, and then determining the final drug concentration for drug sensitivity based on the clinical dosage of the drug.
[0075] Step 3, Drug Sensitivity Testing: Tumor organoids for drug sensitivity testing: Observe the target wells of the 96-well plate under a microscope to understand their current growth status; aspirate and discard the culture medium from the target wells; add an appropriate volume of organoid culture medium to each well. Use a Nikon microscope to photograph the organoids in the 96-well plate to record the morphology of each organoid and save the images;
[0076] Step 4, Fluorescent Staining: On day 0 (D0) after drug administration, add fluorescent antibody (PanCK / EpCAM) and incubate at room temperature for 30 minutes. After staining, aspirate the culture medium and staining solution. Take fluorescence photographs using a fluorescence microscope.
[0077] Step 5: Wash several times with PBS solution, add organoid culture medium and anti-tumor drugs, and continue culturing for 4 days;
[0078] Step 6, Day 4 after drug administration (D4), observe the drug sensitivity samples. Use a Nikon microscope to photograph the organoids in the 96-well plate. The photographed area is the same as the area photographed by Drug0. This is used to record the morphological changes of the same organoid in each well before and after drug administration.
[0079] Step 7: Add fluorescent antibody and dye (PanCK / EpCAM / Calcein-AM / PI), and incubate at room temperature for 30 minutes. After staining, aspirate the culture medium and dye solution, and take fluorescence photographs using a fluorescence microscope.
[0080] Step 8, Data Analysis
[0081] n represents the number of organoids ranging from 30µm to 150µm in each pore;
[0082] For each organ in well D0, ImageJ software measured the major diameter L0. n Area S0 n Observation of transmittance T0 n Surface smoothness R0 n Morphological integrity C0 n .
[0083] For each organ in well D4, ImageJ software measured the long diameter L4. n Area S4 n Observation of transmittance T4 n Surface smoothness R4 n Morphological integrity C4 n And the average fluorescence intensity Fg within the area of organoids determined by live and dead fluorescence staining. n and F rn .
[0084] First, based on the fluorescence image range delineated by the organoid area, the viability of various organs shown by fluorescence staining on D4 is given:
[0085] A4 n =Fg n / (Fg n +Fr n )
[0086] For various organs of D4, after combining and normalizing their morphological observation indicators, the following are observed:
[0087]
[0088] Tb, Rb, and Cb are the baseline transmittance, baseline surface smoothness, and baseline organoid morphological integrity, calibrated by well-formed organoids observed under the same optical parameters.
[0089] There is a relationship between the normalized parameters of morphological indices and fluorescence display activity:
[0090]
[0091] Right now:
[0092] c(M4 n ) = A4 n
[0093] For ease of explanation, the functional relationship is simplified as follows:
[0094] M4 n *c = A4 n
[0095] To balance accuracy and ease of use, the functional relationships are derived by combining the three organoids with the longest major diameters in the first three pores of each pore:
[0096]
[0097] It should be noted that the normalized parameters of morphological indicators and survival rate are usually not linearly related; on the other hand, since the morphological behavior of organs of different cancer types is different, the relationship between morphological indicators and survival rate of organs of each cancer type needs to be fitted and calibrated based on the data of their own cancer type.
[0098] Similarly, for all organs of D0, we can obtain:
[0099]
[0100] And obtain the D0 survival rate:
[0101] A0 n =c(M0) n )
[0102] The scores for each type of organoid are as follows:
[0103] P = L * A
[0104] The changes in the normalized scores of organoids for each well in D0 and D4 are as follows:
[0105]
[0106] Among them, P and P control These represent the sum of organoid scores for the drug administration well and the control well, respectively.
[0107] The results of the control group experiment are shown below Figure 5 The results of the everolimus treatment group are shown in [link to study]. Figure 6 The tumor inhibition rate obtained using the above detection method is shown in the figure. Figure 7 .
[0108] Example 2
[0109] Similar to Example 1, the tumor organoids are nasopharyngeal malignant tumor organoids.
[0110] The results of the control group experiment are shown below Figure 8 The results of the experiment in the gemcitabine + docetaxel group are shown in [link to study]. Figure 9 The experimental results of the vincristine + actinomycin + cyclophosphamide treatment group are shown in [the table below]. Figure 10 The tumor inhibition rate obtained using the above detection method is shown in the figure. Figure 11 It can be seen that the method of the present invention can more effectively discover that the vincristine + actinomycin + cyclophosphamide treatment group has a higher tumor inhibition rate, indicating that the method of the present invention can effectively characterize the tumor inhibition rate of different treatment groups.
[0111] Example 3
[0112] Similar to Example 1, the tumor organoids are intestinal cancer organoids.
[0113] The results of the control group experiment are shown below Figure 12 The results of the regorafenib + sintilimab treatment group are shown in [link to study]. Figure 13 The experimental results of the TAS102 treatment group are shown in [link to experimental results]. Figure 14 The tumor inhibition rate obtained using the above detection method is shown in the figure. Figure 15 It can be seen that the method of the present invention can effectively characterize the tumor inhibition rate of different drug groups.
[0114] Example 4
[0115] Similar to Example 1, the tumor organoids are oropharyngeal malignant tumor organoids.
[0116] The results of the control group experiment are shown below Figure 16 The results of the everolimus treatment group are shown in [link to study]. Figure 17 The results of the palbociclib + apeliximab treatment group are shown in [link to study]. Figure 18 The tumor inhibition rate obtained using the above detection method is shown in the figure. Figure 19 It can be seen that the method of the present invention can effectively characterize the tumor inhibition rate of different drug groups.
[0117] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for detecting drug sensitivity in primary tumor organoids, characterized in that, The drug sensitivity testing method includes: Obtain the major diameter L0 of each organ in well D0. n Area S0 n Observation of transmittance T0 n Surface smoothness R0 n Morphological integrity C0 n ; Obtain the major diameter L4 of each organ in well D4. n Area S4 n Observation of transmittance T4 n Surface smoothness R4 n Morphological integrity C4 n And the average fluorescence intensity Fg within the area of organoids determined by live and dead fluorescence staining. n and Fr n ; Calculate the normalized score change values for each organoid corresponding to wells D0 and D4 using the following formula: in, n represents the number of organoids ranging from 30µm to 150µm in each pore; P and P control These represent the sum of organoid scores for the drug administration well and the control well, respectively. L4 n D4 represents the major diameter of each organ in each well, and A4 represents the major diameter of each organ in each well. n Viability of various organs in each well of D4 as shown by fluorescent staining; L0 n Let D0 be the major diameter of each organ in each orifice, and A0 be the minor diameter of each organ in each orifice. n The viability of each organ in each well of D0 was calculated based on morphological parameters; A4 n The viability of each organ in well D4 as indicated by fluorescent staining was calculated using the following formula: A0 n The following was calculated by fitting a function to morphological parameters and fluorescence display activity: Among them, M0 n The calculation formula is as follows: Among them, T0 n Transmittance and R0 were observed for each organ in D0 well. n For D0, the smoothness of the outer curved surface of each organ in each well, C0 n D0 represents the morphological integrity of various organs in each well; Tb, Rb, and Cb are the baseline observed transmittance, baseline outer surface smoothness, and baseline organ morphological integrity, respectively, calibrated by good organoids observed under the same optical parameters. The formula for calculating c is as follows: in, Among them, T4 n Transmittance of various organs was observed in each well of D4, and in R4... n For the smoothness of the outer curved surface of each organ in D4, C4 n The integrity of the morphology of various organs in each well of D4.
2. The method for detecting drug sensitivity of primary tumor organoids according to claim 1, characterized in that, The method for obtaining the major diameter L0 of various organs in each well of D0. n Area S0 n Observation of transmittance T0 n Surface smoothness R0 n Morphological integrity C0 n ,include: Add the well-grown organoids from the well plate to the culture medium; Add fluorescent antibody and incubate for staining at room temperature; After staining, the culture medium and staining solution were aspirated, and fluorescence images were taken using a fluorescence microscope to obtain the major diameter L0 of each organ in each well (D0). n Area S0 n Observation of transmittance T0 n Surface smoothness R0 n Morphological integrity C0 n .
3. The method for detecting drug sensitivity of primary tumor organoids according to claim 1, characterized in that, The major diameter L4 of each organ in D4 well was obtained. n Area S4 n Observation of transmittance T4 n Surface smoothness R4 n Morphological integrity C4 n And the average fluorescence intensity Fg within the area of organoids determined by live and dead fluorescence staining. n and Fr n ; include: The well-grown organoids in the well plate were added to the culture medium and anti-tumor drugs and cultured for 4 days. Add fluorescent antibody and incubate for staining at room temperature; After staining, the culture medium and staining solution were aspirated, and fluorescence images were taken using a fluorescence microscope to obtain the major diameter L4 of each organ in well D4. n Area S4 n Observation of transmittance T4 n Surface smoothness R4 n Morphological integrity C4 n And the average fluorescence intensity Fg within the area of organoids determined by live and dead fluorescence staining. n and Fr n .
4. The method for detecting drug sensitivity of primary tumor organoids according to claim 1, characterized in that, The drug sensitivity testing method also includes a primary tumor organoid culture step: Wash tumor tissue specimens with pre-cooled cleaning solution; Add digestive fluid and incubate for 30-120 minutes; Add serum-containing PBS to stop digestion, centrifuge, and discard the supernatant. Add DNaseI; centrifuge, resuspend cells and filter using a filter membrane, discard the supernatant after centrifugation; The cell pellet was then treated with erythrocyte lysis buffer. Transfer the resuspended cell suspension into EP tubes; Wash with pre-cooled cleaning solution, centrifuge, add an appropriate amount of matrix gel and mix well, then dispense and plate the material, add organoid culture medium and culture to obtain primary tumor organoids.
5. The method for detecting drug sensitivity of primary tumor organoids according to claim 1, characterized in that, The tumor tissue of the tumor organoids is selected from any one of the following: colon cancer, rectal cancer, gastric cancer, esophageal cancer, oral cancer, osteosarcoma, prostate cancer, breast cancer, brain cancer, neuroendocrine tumors, liver cancer, kidney cancer, pancreatic cancer, lung cancer, melanoma, multiple myeloma, ovarian cancer, cervical cancer, skin cancer, duodenal cancer, gallbladder cancer, intrahepatic and extrahepatic bile duct cancer, head and neck squamous cell carcinoma, synovial sarcoma, liposarcoma, fibrosarcoma, pleomorphic undifferentiated sarcoma, laryngeal papilloma, endometrial cancer, hemangioendothelioma, bladder cancer, ureteral tumors, adrenal tumors, and thyroid cancer tissues.
6. A biochip, wherein the chip performs the drug sensitivity detection method for primary tumor organoids as described in any one of claims 1-5.