Method for analyzing dynamic evolution of tumor nucleolus based on double-thymine block synchronization

Abstract

The application discloses a tumor nucleolus dynamic evolution analysis method based on double-thymine blocking synchronization, which comprises the following steps: culturing the cells to be detected of an ex vivo biopsy sample and reference cells with a deoxycytidine kinase gene knocked out in multiple culture holes; sequentially applying a first blocking, an intermediate release period, a second blocking and a terminal release period to form multiple gradient units with different total blocking exposure amounts, each gradient unit comprising a conventional group culture hole and a saturated rescue group culture hole, and additionally setting a no-blocking control hole and a deoxycytidine-only control hole; measuring a nucleolus disintegration half-time, and inversely calculating the actual total blocking exposure amount of each hole by the reference cells; performing a constrained joint regression, and determining that decoupling is established and outputting a parameter set when the 95% confidence interval of the residual exposure response slope after rescue falls into a preset equivalent interval as a whole. The application separates the synchronization-induced component and the intrinsic component from each other, and the output parameters are comparable between different blocking conditions and batches.

Description

Technical Field

[0001] This invention relates to the field of cell DNA alignment, particularly to the field of cell cycle synchronization and microscopic imaging analysis technology, specifically to a method for dynamic evolution analysis of tumor cell nucleoli based on thymine blocking synchronization. Background Technology

[0002] The size, number, and morphology of the nucleolus have long been used as morphological indicators of the proliferative activity of tumor cells. The disintegration of the nucleolus during prophase I mitosis is a process with a measurable rate, which is related to ribosome biogenetic load and carries information that is difficult to obtain from static indicators. Obtaining this rate requires a sufficient number of cells to enter prophase I within the same observation window.

[0003] Synchronization by thymine blockade is a common method for obtaining this window. Published techniques show that excess thymine deoxynucleotides accumulate as deoxythymidine triphosphate (DTTP) after phosphorylation by thymidine kinase. DTTP binds to the substrate-specific site of the large subunit of ribonucleotide reductase and alters its substrate preference, leading to depletion of the DTTP pool and cell arrest at the G1-S phase boundary. DTTP can be replenished by deoxycytidine kinase via the salvage pathway. The method of labeling nucleolar dense fibrous components with nucleolar fibrous protein and quantifying nucleolar morphology through image segmentation has also been reported.

[0004] Combining the above methods directly still presents unavoidable difficulties: nucleotide pool imbalance triggers replication stress, which acts on the nucleolus, causing a component of inhibition-induced stress that increases with the intensity of inhibition to be mixed into the measured nucleolus disintegration half-time. This component superimposed on the intrinsic component, making it impossible to separate the two in a single measurement under a single inhibition condition. Changing the inhibition concentration or duration yields different results. While canceling the inhibition can avoid this component, the number of cells entering division decreases sharply. The actual exposure of each culture well can also deviate from the set value due to sample loading deviation and evaporation, and current practices still use the set value as the independent variable. The phototoxicity of long-term fluorescence imaging can also prolong the cell cycle, and frame-by-frame resegmentation mixes the shrinkage of the segmentation boundary into the intensity decrease. Summary of the Invention

[0005] This invention provides a method for analyzing the dynamic evolution of tumor cell nucleoli based on thymine blocking synchronization, which addresses the problems of nucleoli alterations induced by synchronization operation being mixed with intrinsic nucleoli dynamics of tumor cells, lack of comparability of measurement results across blocking conditions and across batches, and deviations of the actual exposure amount borne by each culture well from the set value.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for analyzing the dynamic evolution of tumor cell nucleoli synchronized by thymine blockade includes the following steps: Step 1: The test cells and internal control cells taken from the biopsy sample that has been excised are co-cultured in multiple culture wells. The internal control cells are tumor cell lines that have been knocked out of the deoxycytidine kinase gene and whose nucleolar fluorescent labeling is distinguishable from the spectrum of the test cells. Step 2: Apply the first blockade, intermediate release phase, second blockade, and final release phase sequentially to multiple culture wells. The first and second blockades are both performed with thymidine deoxyriboside. The multiple culture wells constitute multiple gradient units with different total blockade exposures. Each gradient unit includes a conventional group culture well and a saturated rescue group culture well. The conventional group culture wells use conventional culture medium during the intermediate and final release phases. The saturated rescue group culture wells are treated with a saturated rescue concentration of deoxycytidine during the intermediate and final release phases. Additionally, there are no blockade control wells (without thymidine deoxyriboside) and deoxycytidine control wells. Step 3: Acquire multi-channel time-series images for multiple culture wells, and measure the nucleolar disintegration half-time of the test cells and internal control cells respectively. The actual total blocking exposure of each culture well is then inverted from the nucleolar disintegration half-time of the internal control cells. The nucleolar disintegration half-time obtained from the unblocked control well is recorded as the reference nucleolar disintegration half-time. Step 4: Using the actual total blocking exposure as the independent variable, and the unblocked control well and the deoxycytidine control well as the anchor point with a total blocking exposure of 0, perform constrained joint regression on the nucleolar disintegration half-time of the conventional group culture well and the saturated salvage group culture well to obtain the intrinsic nucleolar disintegration half-time, artifact sensitivity, salvage operation constant component and salvage residual exposure response slope. When the 95% confidence interval of the salvage residual exposure response slope falls entirely into the preset equivalent interval, it is determined that the blocking induction component decoupling is established. Step 5: Output a set of nucleolar dynamic evolution parameters, including intrinsic nucleolar disintegration half-time, artifact sensitivity, and apparent salvage ratio.

[0007] Further: In step 2, the first blocking exposure is the product of the thymidine deoxyribonucleoside concentration and the first blocking duration; the second blocking exposure is the product of the thymidine deoxyribonucleoside concentration and the second blocking duration; the total blocking exposure is the sum of the first and second blocking exposures. The thymidine deoxyribonucleoside concentration is the same in both the first and second blocking. The thymidine deoxyribonucleoside concentration is set at three levels: 1.0, 1.5, and 2.0 mmol / L. The first blocking duration is 18 hours, with an intermediate release period of 9 hours. The second blocking duration is set at three levels: 12, 15, and 18 hours. The combination of thymidine deoxyribonucleoside concentration and second blocking duration constitutes nine gradient units, with the total blocking exposure ranging from 30 to 72 mmol / L. Step 2 also includes a validation unit. The first blocking duration of the validation unit differs from the first blocking duration of the nine gradient units, and the total blocking exposure of the validation unit is the same as that of one of the gradient units. Three independent replicates are set for each treatment condition.

[0008] Furthermore, before the constrained joint regression in step 4, the nucleolar disintegration half-time of the conventional group culture wells and the calibration unit is used as the observation. An independent coefficient model containing the first blocking exposure coefficient and the second blocking exposure coefficient is fitted. When the 95% confidence interval of the difference between the two coefficients falls into the preset equivalent interval, the first blocking exposure and the second blocking exposure are combined into a total blocking exposure for participation in the constrained joint regression. When the gradient unit with the same blocking exposure as the calibration unit falls into the equivalent interval of ±5% of the reference nucleolar disintegration half-time of the difference obtained from the conventional group culture wells in the 95% confidence interval of the difference between the nucleolar disintegration half-time of the calibration unit and the total blocking exposure, the definition of the total blocking exposure is deemed appropriate.

[0009] Furthermore: In step 2, pre-rescue group culture wells and post-rescue group culture wells are set up in the three gradient units with the lowest, middle and highest total blocking exposure. The pre-rescue group culture wells are only given a saturated rescue concentration of deoxycytidine during the intermediate release phase, and the post-rescue group culture wells are only given a saturated rescue concentration of deoxycytidine during the final release phase. Step 4 also includes calculating the first segment difference, the second segment difference and the total difference in the three gradient units respectively. The first segment difference, the second segment difference and the total difference are the differences in nucleolar disintegration half-time obtained by the conventional group culture well and the pre-rescue group culture well, the post-rescue group culture well and the saturated rescue group culture well, respectively. When the 95% confidence interval of the value obtained by subtracting the total difference from the sum of the first segment difference and the second segment difference falls within the equivalent interval of ±10% of the reference nucleolar disintegration half-time in all three gradient units, it is determined that the blocking induction components contributed by the first blocking and the second blocking satisfy the additivity.

[0010] Furthermore: The saturation salvage concentration in step 2 was determined by the gradient unit condition of the preliminary experiment with the highest total blocking exposure. The deoxycytidine concentration in the preliminary experiment was set at 5 levels of 0, 8, 24, 48 and 96 μmol / L. The lowest deoxycytidine concentration that fell within the equivalent range of ±5% of the half-time difference of nucleolar disintegration obtained from the difference of two adjacent deoxycytidine concentrations was taken as the saturation salvage concentration. The saturation salvage concentration was used uniformly for all culture wells in step 2 where deoxycytidine was applied.

[0011] Further: In step 1, the test cells were obtained from ex vivo biopsy samples through enzymatic digestion, primary culture, and tumor origin identification. An enhanced green fluorescent protein coding sequence was knocked into the nucleolar fibroin intrinsic site using homologous recombination. The internal control cells had a monomeric red fluorescent protein coding sequence knocked into the nucleolar fibroin intrinsic site. The internal control cells and test cells were co-cultured at a seeding density of 1:9. The 95% confidence interval of the difference between the nucleolar disintegration half-time obtained from co-cultured wells and individually cultured wells fell within the equivalent interval of ±5% of the reference nucleolar disintegration half-time. The calibration intercept and calibration exposure response slope of the internal control cells were given by a single calibration experiment. In step 3, the actual total blocking exposure was retrieved by taking the difference between the median nucleolar disintegration half-time of the internal control cells in the culture wells and the calibration intercept, and dividing it by the calibration exposure response slope.

[0012] Further: In step 3, an automated high-content live-cell imaging device was used to independently time and collect images according to the start time of the terminal release phase of each culture well. The acquisition included inspection acquisition and burst acquisition. Inspection acquisition, from 6 to 16 hours after the start of the terminal release phase, only collected images of chromatin channels labeled with chromatin fluorescent dye at 10-minute intervals. When the standard deviation of grayscale in the nucleus region reached 1.5 times or more of the average value obtained from the previous 6 rounds of inspection acquisition for the same cell, the cell was determined to have entered the prophase, and the corresponding acquisition round was recorded as the prophase determination round, and the corresponding field of view was switched to burst acquisition. Burst acquisition lasted for 90 minutes at 2-minute intervals, and simultaneously acquired images of the nucleolar channel of the cell under test, the nucleolar channel of the internal reference cell, and the chromatin channel. The total time of a single round of 3-channel scanning was within 2 minutes, and the cumulative irradiation of a single cell was within 8 joules per square centimeter.

[0013] Further: The method for determining the nucleolar disintegration half-time in step 3 includes: obtaining a fixed measurement mask by thresholding and morphological dilation of the nucleolar channel images from the previous determination rounds; estimating the translational and rotational components round by round from the centroid and principal axis of the second central moment of the nucleus region in the chromatin channel, and applying the same rigid body transformation to the fixed measurement mask; quotienting the integral intensity within the fixed measurement mask to a bleaching reference to obtain the normalized integral intensity, where the bleaching reference is the round-by-round mean of the integral intensity of cells in interphase within the same size mask in the same field of view; and fitting a 4-parameter logistic function to the time series of the normalized integral intensity using the previous determination round as the time origin. The high plateau before disintegration and the low plateau after disintegration are obtained. The half-time of nucleolus disintegration is the difference between the time when the normalized integral intensity reaches 50% of the amplitude between the high plateau before disintegration and the low plateau after disintegration and the time origin. The complete trajectory is the trajectory with a fitting determination coefficient of 0.95 or higher, the difference between the high plateau before disintegration and the low plateau after disintegration being 5 times or more of the standard deviation of the reference noise within the fixed measurement mask, and the cell being within the field of view throughout the burst acquisition. For cells that remain in the high plateau region before disintegration during burst acquisition, the time at the end of burst acquisition is used as the right censored observation. The right censored observation and the complete trajectory are included in the constrained joint regression of step 4 along with the log-normal accelerated failure time likelihood.

[0014] Further: In step 2, parallel quality control cultures and light and dye control wells were set up. The parallel quality control cultures were cultured separately from multiple culture wells under the same treatment conditions. A portion of the parallel quality control cultures were pulse-labeled with thymidine analogs at the beginning of the terminal release phase. After fixation, dual-parameter flow cytometry was performed to measure the thymidine analog incorporation and DNA content. The synchronization criterion was that the proportion of early S-phase cells with diploid DNA content and positive thymidine analog incorporation reached 75% or more. Another portion of the parallel quality control cultures were subjected to liquid chromatography-tandem mass spectrometry to determine the ratio of deoxycytidine triphosphate to deoxythymidine triphosphate 1 hour after the start of the terminal release phase. The ratio of deoxycytidine triphosphate to deoxythymidine triphosphate was used as the saturation salvage group culture wells. The correction criterion is that the deviation of the content ratio of the corresponding well from the content ratio of the unblocked control well is within 20%. The mitotic entry rate of the light and dye control wells is measured under three conditions: burst acquisition with chromatin fluorescent dye applied, bright-field inspection acquisition with chromatin fluorescent dye applied, and bright-field inspection acquisition without chromatin fluorescent dye. Bright-field inspection acquisition is inspection acquisition performed by replacing fluorescence excitation with transmitted illumination. The criterion is that the 95% confidence interval of the difference between the mitotic entry rates obtained under any two conditions falls within the equivalent interval of ±3 percentage points. The criterion is that the influence of light and dye is acceptable. When the synchronization criterion, the correction criterion, and the criterion of the acceptable influence of light and dye are all met, step 3 is continued.

[0015] Further: In step 4, the preset equivalent interval uses the equivalent boundary value as the positive and negative boundary. The equivalent boundary value is 10% of the reference nucleolar disintegration half-time divided by the difference between the maximum and minimum total blocking exposure values ​​in multiple gradient units. Constrained joint regression ensures that the nucleolar disintegration half-time of the conventional group culture wells is equal to the sum of the products of the intrinsic nucleolar disintegration half-time, artifact sensitivity, and actual total blocking exposure. It also ensures that the nucleolar disintegration half-time of the saturated salvage group culture wells is equal to the sum of the products of the intrinsic nucleolar disintegration half-time, the salvage operation constant component, the salvage residual exposure response slope, and the actual total blocking exposure. The salvage operation constant component is the difference between the nucleolar disintegration half-time obtained from the deoxycytidine single control well and the non-blocking control well. The apparent salvage ratio is the difference between 1 and the ratio of the salvage residual exposure response slope to the artifact sensitivity. The blocking induction component is the nucleolar disintegration half-time that varies with the actual exposure value. The total blocking exposure change component; constrained joint regression with single-cell nucleolar disintegration half-time as the observation and culture wells as the random effect layer; the conditions for determining the decoupling of the blocking-induced component also include the lower limit of the 95% confidence interval of the artifact sensitivity reaching 3 times or more of the equivalent threshold, the lower limit of the 95% confidence interval of the apparent salvage ratio reaching 0.9 or more, and the 95% confidence interval of the difference between the mean value of the nucleolar disintegration half-time obtained from multiple gradient units of the saturated salvage group culture wells minus the salvage operation constant component and the reference nucleolar disintegration half-time falling within the equivalent interval of ±10% of the reference nucleolar disintegration half-time; the nucleolar dynamic evolution parameter set output in step 5 also includes the salvage operation constant component, mitotic entry rate, right censoring observation ratio, cell death rate, and the coefficient of variation of intrinsic nucleolar disintegration half-time among 3 independent batches.

[0016] By employing the above technical solution, the present invention has the following beneficial effects: It separates the component induced by synchronization operation from the component carried by tumor cells themselves on the same sample, ensuring that the output parameters remain consistent across different blocking concentrations, blocking durations, and batches; it uses deoxycytidine to salvage the exposure response slope into a pre-set equivalent range—an achievable and verifiable state—replacing the experimentally unattainable method of extrapolating to a zero-exposure point, thus covering the components of both blocking phases; and it uses deoxycytidine kinase gene knockout internal control cells as well-dose dosimeters, transforming the independent variable written according to the set value into… The measured values, obtained well by well, incorporate sampling bias and well-to-well differences caused by evaporation into the measured values ​​of the independent variables, reducing inter-well and inter-batch dispersion. The additivity test, constructed through segmented salvage, provides a preliminary verification for component decomposition, ensuring that the decoupling conclusions have a verifiable basis. Event-triggered two-stage acquisition concentrates light dose on the information-carrying time period, making cell cycle duration largely unaffected by observation. A fixed measurement mask, combined with rigid registration and bleaching reference, isolates segmentation boundary drift and fluorescence bleaching from intensity reduction. The estimation method, including right-censored observations, allows cells entering division later to participate in the estimation, suppressing selection bias caused by retaining only those that have completed division. These effects provide a basis for using the output parameters as quantitative indicators. Attached Figure Description

[0017] Figure 1 This is a timing diagram illustrating the synchronization of thymidine blockade and segmented deoxycytidine salvage in an embodiment of the present invention.

[0018] Figure 2 This is a schematic diagram illustrating the principle of deoxyribonucleoside triphosphate pool imbalance and deoxycytidine rescue pathway in embodiments of the present invention.

[0019] Figure 3 This is a schematic diagram of the two-dimensional factorial gradient unit distribution and the isotopic blocking exposure line in an embodiment of the present invention.

[0020] Figure 4 This is a schematic diagram illustrating the concentration-response relationship in the saturation salvage concentration determination process of this invention.

[0021] Figure 5 This is a schematic diagram illustrating the rigid body registration principle of the fixed measurement mask as it translates and rotates with the cell nucleus in an embodiment of the present invention.

[0022] Figure 6 This is a schematic diagram illustrating the principle of normalized integral intensity fitting and nucleolus disintegration half-time definition in this embodiment of the invention.

[0023] Figure 7 This is a schematic diagram illustrating the principle of inversion between internal control cell calibration and actual total blocking exposure in this embodiment of the invention.

[0024] Figure 8 This is a schematic diagram illustrating the principle of constrained joint regression dual-branch and exposure independence criterion in an embodiment of the present invention.

[0025] Figure 9 This is a schematic diagram illustrating the principle of the piecewise salvage additivity test in an embodiment of the present invention. Detailed Implementation

[0026] A method for analyzing the dynamic evolution of tumor cell nucleoli synchronized by thymine blockade includes the following steps: Step 1: The test cells and internal control cells taken from the biopsy sample that has been excised are co-cultured in multiple culture wells. The internal control cells are tumor cell lines that have been knocked out of the deoxycytidine kinase gene and whose nucleolar fluorescent labeling is distinguishable from the spectrum of the test cells. Step 2: Apply the first blockade, intermediate release phase, second blockade, and final release phase sequentially to multiple culture wells. The first and second blockades are both performed with thymidine deoxyriboside. The multiple culture wells constitute multiple gradient units with different total blockade exposures. Each gradient unit includes a conventional group culture well and a saturated rescue group culture well. The conventional group culture wells use conventional culture medium during the intermediate and final release phases. The saturated rescue group culture wells are treated with a saturated rescue concentration of deoxycytidine during the intermediate and final release phases. Additionally, there are no blockade control wells (without thymidine deoxyriboside) and deoxycytidine control wells. Step 3: Acquire multi-channel time-series images for multiple culture wells, and measure the nucleolar disintegration half-time of the test cells and internal control cells respectively. The actual total blocking exposure of each culture well is then inverted from the nucleolar disintegration half-time of the internal control cells. The nucleolar disintegration half-time obtained from the unblocked control well is recorded as the reference nucleolar disintegration half-time. Step 4: Using the actual total blocking exposure as the independent variable, and the unblocked control well and the deoxycytidine control well as the anchor point with a total blocking exposure of 0, perform constrained joint regression on the nucleolar disintegration half-time of the conventional group culture well and the saturated salvage group culture well to obtain the intrinsic nucleolar disintegration half-time, artifact sensitivity, salvage operation constant component and salvage residual exposure response slope. When the 95% confidence interval of the salvage residual exposure response slope falls entirely into the preset equivalent interval, it is determined that the blocking induction component decoupling is established. Step 5: Output a set of nucleolar dynamic evolution parameters, including intrinsic nucleolar disintegration half-time, artifact sensitivity, and apparent salvage ratio.

[0027] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. The terminology used in the following description is consistent with the foregoing technical solutions. Unless otherwise specified, culture, passage, and fixation operations are performed according to conventional cell biology methods.

[0028] To obtain a sufficient number of promitotic cells within a controlled observation window, thymine-induced synchronization must be applied. However, the nucleotide pool imbalance upon which synchronization depends alters the nucleolar disintegration kinetics, introducing a component induced by the blocking operation into the measured nucleolar disintegration half-time. Existing methods mitigate this component by reducing the blocking concentration or shortening the blocking duration, but this weakens the synchronization effect and reduces the number of effective trajectories within the observation window, compromising both throughput and accuracy. This invention constructs a set of conditions with varying exposure levels on the same sample and utilizes biochemical methods capable of specifically addressing nucleotide pool imbalances, making the magnitude of the blocking-induced component an actively adjustable and directly observable quantity.

[0029] Suppose that the measured nucleolar disintegration half-time is the superposition of the intrinsic nucleolar disintegration half-time and the blocking-induced component, the latter monotonically increasing with the total blocking exposure. An intuitive approach is to regress the measured value against the total blocking exposure and extrapolate it to a point where the exposure is zero, using the intercept as the intrinsic value. This approach has two drawbacks. A zero exposure means abandoning synchronization, a point that is experimentally unattainable, and the extrapolated result lacks independent observations for comparison. Furthermore, if the gradient is applied only to the second blocking, the exposure from the first blocking remains constant under all conditions, and the extrapolated intercept still contains the contribution from the first blocking, which does not disappear as the exposure from the second blocking approaches zero. Based on this, this invention makes two adjustments: defining the total blocking exposure as the sum of the first and second blocking exposures, and making both change simultaneously with the gradient; and applying deoxycytidine salvage to the intermediate and final release phases to cover the components left by each of the two blocking phases. With this adjustment, the criterion is changed from the intercept obtained by extrapolation to whether the regression slope of the measured value under saturation salvage conditions with respect to the total blocking exposure falls into the preset equivalent interval. This state can be achieved experimentally and can be tested one by one using statistical methods.

[0030] Figure 1 This illustration demonstrates the timing principle of thymine blockade synchronization and segmented deoxycytidine rescue in an embodiment of the present invention. Starting from the beginning of the time axis, each culture well sequentially experiences the first blockade, intermediate release phase, second blockade, and final release phase. The segments filled with diagonal lines in the figure represent the two blockades applied with thymine deoxynucleotides, while the blank segments represent the two release phases. The four parallel branches correspond to the conventional group culture wells, the saturated rescue group culture wells, the pre-rescue group culture wells, and the post-rescue group culture wells, respectively. The solid black bands above each branch mark the time period for deoxycytidine application: the conventional group culture wells use conventional culture medium for both release phases, and there is no solid band above them; the saturated rescue group culture wells receive deoxycytidine in both the intermediate and final release phases; the pre-rescue group culture wells receive it only in the intermediate release phase; and the post-rescue group culture wells receive it only in the final release phase. Figure 1 The image acquisition branch below shows the time span of the inspection acquisition and the burst acquisition periods triggered within it. Figure 1 The blocking parameters of the four branches are exactly the same, and the only difference between them is which release phase the deoxycytidine is applied to. This design makes the subsequent fractional decomposition have a clear experimental correspondence.

[0031] The cells to be tested were prepared as follows: Ex vivo biopsy samples were taken, minced, and digested with a collagenase-containing digestive solution at 37°C for 40 to 90 minutes. After filtration through a 70-micron sieve to remove red blood cells, the cells were inoculated into a culture medium containing 10% fetal bovine serum for primary culture. The tumor origin was confirmed by pathological morphology and immunocytochemistry. Subsequently, an enhanced green fluorescent protein (GFP) coding sequence was knocked into the endogenous site of nucleolar fibrin via homologous recombination. Transient plasmid transfection or lentiviral overexpression was not used here because the nucleolus is a non-membrane structure, and the concentration of its internal components directly affects phase separation behavior. Exogenous overexpression would increase the abundance of nucleolar fibrin and alter the nucleolus size and disintegration kinetics, causing the measured nucleolar disintegration half-time to deviate from the true state of the sample. To verify whether the label itself introduces bias, labeled cells and unlabeled cells from the same batch were fixed and stained with silver nitrate. The median area of ​​the nucleolar silver-stained region was compared between the two. The requirement was that the 95% confidence interval of the difference between the two should fall within an equivalent interval of ±10% of the median area of ​​the nucleolar silver-stained region. This step aims to confirm that the two are sufficiently close, but the significance test cannot provide a conclusion of closeness; therefore, the equivalent interval is used.

[0032] The internal control cells were obtained from a stable tumor cell line with a clearly defined nucleolus. The deoxycytidine kinase gene was first knocked out using gene editing, and then a monomeric red fluorescent protein coding sequence was knocked into the endogenous nucleolar fibrin site. The selection of deoxycytidine kinase knockout was based on the following criteria: Figure 2 The diagram shows the nucleotide metabolism relationships.

[0033] Figure 2 This illustrates the mechanism of deoxynucleotide triphosphate pool imbalance and the deoxycytidine salvage pathway. Thymidine deoxynucleotides are phosphorylated by thymidine kinase and progressively converted to deoxythymidine triphosphate. Deoxythymidine triphosphate acts allosterically on ribonucleotide reductase, as indicated by the broken line with a horizontal bar in the figure. The consequence is a decrease in the reduction rate of cytidine diphosphate to deoxycytidine diphosphate, leading to depletion of the deoxycytidine triphosphate pool. This depletion then forms the blocking-inducing component in the nucleolar disintegration half-time through the replication stress pathway. Figure 2 The salvage pathway below shows that deoxycytidine is phosphorylated to deoxycytidine monophosphate by deoxycytidine kinase, and then deoxycytidine diphosphate is generated by deoxycytidine monophosphate kinase and merged into the same node, bypassing the inhibited ribonucleotide reductase and directly replenishing the deoxycytidine triphosphate pool. Figure 2The cross on the deoxycytidine kinase catalytic step indicates that this step was knocked out in the internal control cells. The response of the internal control cells to thymidine deoxyribonucleoside exposure is mediated by thymidine kinase, and therefore this response is fully preserved; however, the compensatory effect of deoxycytidine on the internal control cells is truncated, and its apparent salvage ratio is in the equivalent zero range after a single calibration. Therefore, the internal control cells respond to the exposure level with the same relationship in both the standard group and the saturated salvage group wells, and can act as an in-well exposure dosimeter, transforming the independent variable, which was originally only writeable according to the sample loading setting, into a quantity that can be measured in every culture well.

[0034] Internal control cells and test cells were co-cultured in the same wells at a seeding density of 1:9. Setting the seeding density of internal control cells to one-ninth that of test cells, rather than the same amount, was to minimize potential paracrine effects and nutrient competition between the two cell types while still obtaining sufficient internal control cell tracks per well. To verify this effect, separate culture wells were set up for internal control cells to be cultured alone. The 95% confidence interval of the difference between the nucleolar disintegration half-times obtained from the co-cultured and the separately cultured wells was required to fall within a 5% equivalent interval of the reference nucleolar disintegration half-time. The reference nucleolar disintegration half-time was obtained from the unblocked control wells and served as a common scale for all equivalent intervals.

[0035] The administration of blocking and rescue was arranged in a two-dimensional factorial manner. Thymine deoxyribonucleotide concentrations were used at three levels: 1.0, 1.5, and 2.0 mmol / L, with the same values ​​used in the first and second blocking phases. The duration of the first blocking phase was 18 hours, with an intermediate release period of 9 hours. The duration of the second blocking phase was used at three levels: 12, 15, and 18 hours. The total combination of concentration and second blocking duration constituted nine gradient units. The exposure levels for the first, second, and total blocking phases were sequentially arranged as follows: In the formula, c represents the concentration of thymidine deoxyribonucleoside in millimoles per liter; t1 represents the duration of the first blockade, t2 represents the duration of the second blockade, both in hours; E1 represents the exposure dose during the first blockade, E2 represents the exposure dose during the second blockade, and E represents the total blockade exposure dose, all in millimoles per hour per liter. Calculated using the above formula, the total blockade exposure doses for the nine gradient units are 30, 33, 36, 45, 49.5, 54, 60, 66, and 72 millimoles per hour per liter, respectively, covering a range of 2.4 times. The concentration-duration product form is used because the accumulation of intracellular deoxythymidine triphosphate during the blockade period is approximately proportional to the extracellular concentration, while the duration of imbalance is proportional to the blockade duration; the product of the two corresponds to the cumulative effect. The appropriateness of this form is verified by the two exposure equivalence tests and validation units described later.

[0036] Figure 3The figure shows the distribution of nine gradient units on the plane formed by the concentration and the second blocking time, as well as the isotopic blocking exposure line. Solid dots in the figure represent the nine gradient units, with their respective total blocking exposure values ​​labeled next to them; dashed lines are isotopic blocking exposure lines, with the values ​​labeled at the ends of the lines representing the total blocking exposure corresponding to that line. Figure 3 The hollow squares in the diagram represent calibration units with a thymidine deoxyribonucleoside concentration of 1.2 mmol / L, a first blocking duration of 12 hours, a second blocking duration of 18 hours, and a total blocking exposure of 36 mmol / L. This is the same as the gradient unit with a concentration of 1.0 mmol / L, a first blocking duration of 18 hours, and a second blocking duration of 18 hours. The two units are connected by a thick vertical line. The first blocking duration of the calibration unit differs from all other units; therefore, under the premise of the same total blocking exposure, the ratio of its first to second blocking exposure differs significantly from that of the compared gradient unit. If the difference between the two nucleolar disintegration half-times falls within the equivalent range of ±5% of the reference nucleolar disintegration half-time, then defining the total blocking exposure as the sum of the products is appropriate, and the effects of the unit exposure from the two blocking events can be calculated together.

[0037] Three independent duplicate wells were set up for each treatment condition, with the well positions randomly arranged on the culture plate to avoid systematic bias between treatment conditions caused by evaporation differences and temperature gradients in the edge wells. The three levels of the second block duration staggered the start time of the terminal release period of each culture well by 3 hours, thus dispersing the culture wells that needed to be acquired into three time groups, keeping the load on the imaging device within a manageable range at any given time.

[0038] The saturation salvage concentration was determined through independent pilot experiments conducted under gradient cell conditions with the highest total blocking exposure. The rationale for choosing the highest exposure level is that the required deoxycytidine dose for recovery increases with the depth of nucleotide pool imbalance; a concentration sufficient to plateau the response at moderate exposure levels may not cover the highest exposure level. If the concentration determined under moderate conditions were generalized to all conditions, the salvage at the highest exposure level would be incomplete. The pilot experiments used five deoxycytidine concentration levels: 0, 8, 24, 48, and 96 μmol / L.

[0039] Figure 4The figure shows the variation of nucleolar disintegration half-time with deoxycytidine concentration during the determination of saturation salvage concentration. The solid dots and error bars in the figure represent the measured values ​​at each concentration; the solid line is the fitted curve; the dotted line is the horizontal reference line extending to the right from each measurement point; and the horizontal dashed line is the reference nucleolar disintegration half-time. With increasing deoxycytidine concentration, the nucleolar disintegration half-time first decreases sharply and then plateaus: decreasing by 5.34 minutes from 0 μmol / L to 8 μmol / L, by 4.12 minutes from 8 μmol / L to 24 μmol / L, by only 0.15 minutes from 24 μmol / L to 48 μmol / L, and by only 0.03 minutes from 48 μmol / L to 96 μmol / L. Figure 4 The vertical double arrow with caps on the right indicates the preset equivalent interval. Centered on the horizontal reference line corresponding to a deoxycytidine concentration of 24 μmol / L, it extends upwards and downwards by 5% of the reference nucleolus disintegration half-time. When the reference nucleolus disintegration half-time is 14.80 minutes, the total width is 1.48 minutes. The horizontal reference lines corresponding to deoxycytidine concentrations of 48 μmol / L and 96 μmol / L both fall between the upper and lower caps of this double arrow, meaning the difference between adjacent concentrations is within the preset equivalent interval. The determination rule is: the lowest deoxycytidine concentration whose 95% confidence interval of the difference between the nucleolus disintegration half-times obtained from two adjacent deoxycytidine concentrations falls within this equivalent interval is taken as the saturation compensation concentration. According to this rule, the difference between 24 μmol / L and 48 μmol / L satisfies this condition; therefore, the saturation compensation concentration is 24 μmol / L. Figure 4 The values ​​are marked with vertical dotted lines. An equivalence test is used here instead of the usual significance test because a confidence interval containing 0 only indicates no difference was detected; insufficient sample size or large fluctuations will also result in a result containing 0. Only when the value falls within the pre-defined equivalence interval does it indicate a negligible difference. All culture wells treated with deoxycytidine used the same saturation salvage concentration. Since this concentration is within the response plateau, the difference between the measured values ​​at the highest exposure in the saturation salvage group wells in the main experiment and the measured values ​​at this concentration in this preliminary experiment is within the measurement error range.

[0040] The degree of synchronization and the replenishment of the nucleotide pool are determined by parallel quality control cultures. Flow cytometry and liquid chromatography-tandem mass spectrometry both require cell fixation or lysis, which are destructive measurements; the tested cells cannot be used for live-cell imaging. Therefore, parallel quality control cultures are cultured separately from the multiple wells used for imaging and are set up under the same treatment conditions. A portion of the parallel quality control cultures are pulse-labeled with thymidine analogs at a concentration of 10 μmol / L for 15 minutes at the start of the terminal release phase. After fixation and click labeling, a two-parameter flow cytometry analysis is performed, measuring both thymidine analog incorporation and DNA content. A synchronization criterion is defined as early S-phase cells with a diploid DNA content and a thymidine analog incorporation positivity rate of 75% or higher. DNA content alone cannot distinguish late G1 phase cells from cells just entering S phase, and thymidine analog incorporation directly indicates the initiation of DNA synthesis; therefore, a two-parameter analysis is used instead of simply using DNA content distribution. Another set of parallel quality control cultures were subjected to liquid chromatography-tandem mass spectrometry to determine the content ratio of deoxycytidine triphosphate to deoxythymidine triphosphate 1 hour after the start of the terminal release period. The content ratio of the saturated salvage group wells was taken as the compensation criterion if the deviation of the content ratio of the corresponding wells of the unblocked control was within 20%. All nine gradient units were required to meet this criterion to confirm that the selected saturated salvage concentration was effective throughout the entire exposure range.

[0041] The effects of light and dye were verified using control wells. The mitotic entry rate was measured in the culture wells under three conditions: burst acquisition with chromatin fluorescent dye applied, bright-field inspection acquisition with chromatin fluorescent dye applied, and bright-field inspection acquisition without chromatin fluorescent dye. Bright-field inspection acquisition used transmitted illumination instead of fluorescence excitation, with an illumination power density within 0.1 watts per square centimeter. The 95% confidence interval of the difference between the mitotic entry rates obtained under any two conditions falling within a ±3 percentage point equivalent interval was used as the acceptable criterion for the effects of light and dye. The endpoint mitotic index only reflects the proportion of cells in a dividing state at a certain moment and cannot distinguish between a decrease in the number of cells entering division and a prolonged division process; therefore, the mitotic entry rate was used as the comparison metric. Image acquisition and nucleolar disintegration half-time measurements were only performed after the synchronization criterion, the complementation criterion, and the acceptable criterion for the effects of light and dye were all met.

[0042] Image acquisition was performed using an automated high-content live-cell imaging system with a 40x objective lens and a numerical aperture of 0.95. The environmental conditions were 37°C and 5% CO2 (volume fraction). Each culture well was independently timed according to its own terminal release phase start time. Acquisition was divided into two phases: inspection acquisition and burst acquisition. Inspection acquisition began 6 hours after the start of the terminal release phase and ended 16 hours later, at 10-minute intervals. Only images of chromatin channels labeled with a chromatin fluorescent dye were acquired. The working concentration of the chromatin fluorescent dye was 50 nanomoles per liter, the excitation wavelength was 640 nm, the exposure time was 25 ms, and the sample surface power density was 0.8 W / cm². During the inspection phase, only chromatin channels were acquired, not nucleolar channels. This is because the inspection task is only to determine when the cell enters prophase, and chromatin condensation is the most robust indicator of this event. The information carried by nucleolar channels is only valuable during the disintegration process; acquiring them prematurely would consume photon budget and exacerbate bleaching.

[0043] The criterion for entering prophase is derived from chromatin channel images: when the standard deviation of grayscale in a certain nuclear region reaches 1.5 times or more the average value obtained from the previous 6 rounds of inspections of the same cell, the cell is determined to have entered prophase. The corresponding inspection round is recorded as the prophase determination round, and the field of view is then switched to burst inspection. Chromatin condensation changes the spatial distribution of intranuclear fluorescence rather than the total amount. The average grayscale remains basically unchanged during condensation, while the standard deviation increases significantly. Therefore, the grayscale standard deviation is used as the trigger value. The 1.5-fold threshold was determined by preliminary experiments: in unsynchronized control cells, the coefficient of variation of the interphase nuclear region grayscale standard deviation fluctuation is within 10%. Taking 1.5 times is equivalent to a deviation of more than 5 times the standard deviation, which avoids false triggering caused by random fluctuations and can trigger early in the chromatin condensation process.

[0044] Burst acquisition was conducted at 2-minute intervals for 90 minutes, simultaneously acquiring images of three channels: the nucleolar channel of the target cell, the nucleolar channel of the internal control cell, and the chromatin channel. The excitation wavelengths for two of the nucleolar channels were 488 nm and 561 nm, respectively, with an exposure time of 50 ms for each and a sample surface power density of 1.2 W / cm². The total duration of a single round of 3-channel scanning was controlled within 2 minutes to ensure consistency between the nominal and actual acquisition intervals. The actual timestamp for each field of view was recorded and used for time axis correction to eliminate timing jitter during the multi-well cycle. Based on these parameters, the cumulative irradiance of a single cell throughout the entire observation process remained within 8 joules / cm². This limit was used to control the light dose within a range that prevented the cell cycle from being prolonged, ensuring that the measured nucleolar disintegration half-times reflected the cell's intrinsic properties rather than being a product of the observation process. The illumination and dye control wells were set to verify this limit.

[0045] The measurement process for nucleolar disintegration half-time is as follows: Nucleolar channel images from previous rounds are thresholded, and connected regions with an area of ​​0.5 square micrometers or larger are selected and morphologically dilated by 3 pixels to obtain a fixed measurement mask. Once determined, this mask maintains its shape and area throughout the burst acquisition process, undergoing only rigid transformations with the cell nucleus. A fixed mask is used instead of re-segmenting round by round because the nucleolar disintegration process is accompanied by a transition of nucleolar fibrin fluorescence signals from an aggregated state to a diffuse state. Connected regions obtained from round-by-round segmentation shrink accordingly, resulting in an indistinguishable difference between the measured intensity decrease and the shrinking measurement area. The fixed mask separates the measurement area from the morphological changes of the measured object, ensuring that the intensity decrease only reflects the actual reduction of fluorescent material within the mask area. The 3-pixel dilation is used to include the transition zone at the nucleolar edge in the mask from the initial round, avoiding underestimation caused by outward diffusion of material in the early stages of disintegration.

[0046] Figure 5 This illustrates the rigid body registration principle of a fixed measurement mask as the nucleus translates and rotates. The dashed ellipse in the diagram represents the nucleus outline, the solid line running through the ellipse is the principal axis determined by the second central moment, the cross mark is the centroid, and the thick solid closed polygon is the fixed measurement mask; the area with filled lines inside the polygon represents the nucleolar region. The three diagrams from left to right correspond to the 1st, kth, and nth rounds of data acquisition, respectively, with principal axis rotation angles of 0 degrees, 18 degrees, and 34 degrees. The shape and area of ​​the mask remain unchanged, moving only as the centroid of the nucleus translates and the principal axis rotates. The principal axis rotation angle is determined by the second central moment of the nucleus region in the chromatin channel, i.e. In the formula, θ is the principal axis rotation angle, and μ is the... 20 With μ 02 These are the second-order central moments of the nuclear region along the transverse and longitudinal directions, respectively, μ. 11 The three moments are mixed second-order central moments, which are all calculated directly from the deviation of the pixel coordinates of the cell nucleus region in the chromatin channel from the centroid according to the definition of moments.

[0047] The coordinates of any point on the mask are transformed from the coordinates of the previous rounds to the coordinates of the k-th round, and the transformation relationship is as follows: , In the formula, x and y are the coordinates of the point in the previous rounds, and x′ and y′ are its coordinates in the kth round. c With y c To determine the centroid coordinates of the cell nucleus region in the previous round, x c ′ and y c θ' represents the centroid coordinates of the nucleus region in the k-th round. k This represents the difference between the principal axis rotation angle in round k and the principal axis rotation angle in the previous judgment round. Cells entering prophase often undergo overall rotation. If the mask only translates, the nucleolus will gradually move out of the mask range. The resulting decrease in strength is indistinguishable from the disintegration process, so a rotational component is introduced here.

[0048] The effects of fluorescence bleaching were eliminated using a bleaching reference. The mean value of the integrated intensity of cells in interphase within the same field of view and within a mask of the same size was taken as the bleaching reference. The normalized integrated intensity was obtained by quoting the integrated intensity within the fixed measurement mask against the bleaching reference. In the formula, I k For the k-th round of fixed measurement, B is the integral intensity within the mask. k J serves as the bleaching reference for the kth round. k The normalized integral intensity is for the kth round. Interphase cells and the tested cells are subjected to the same lighting history and environmental conditions, and their nucleoli do not disintegrate within the observation window. The change in intensity only reflects bleaching and lighting fluctuations. As a reference obtained locally without the need for additional assumptions, interphase cells from the same field of view are selected instead of a pre-measured bleaching curve.

[0049] Using the previous round as the time origin, a 4-parameter logistic function is fitted to the time series of normalized integral intensity. In the formula, τ is the time from the previous judgment round, in minutes; the function value is the normalized integral intensity at time τ, dimensionless; A1 is the high plateau before disintegration, and A2 is the low plateau after disintegration, both dimensionless; T is the half-time of nucleolar disintegration, in minutes; p is the steepness parameter, dimensionless. According to this formula, the half-time of nucleolar disintegration is the difference between the time when the normalized integral intensity reaches 50% of the amplitude between the high plateau before disintegration and the low plateau after disintegration and the origin of time. The half-time is defined as 50% of the amplitude between the two plateaus rather than half of the initial value because after nucleolar disintegration, not all nucleolar fibrin fluorescence signals disappear from the mask range; a considerable proportion of the signal remains in situ in a diffuse form, and the low plateau after disintegration is significantly higher than 0. If half of the initial value is used as the criterion, the half-time cannot be obtained when the low plateau after disintegration is higher than this value, and the measurement fails on some cells.

[0050] Figure 6 The figure shows the fitting of the normalized integral intensity and the definition of the nucleolus disintegration half-time. The hollow circles in the figure represent the normalized integral intensity measured in each round, the solid lines are the 4-parameter logistic fitting curves, the upper and lower horizontal dashed lines are the high plateau before disintegration and the low plateau after disintegration, respectively, the horizontal dotted line between the two is the level of 50% amplitude, and the x-coordinate of the intersection of the vertical dotted line and the 50% amplitude line is the nucleolus disintegration half-time. Figure 6 The vertical double arrows in the image represent the platform difference. Figure 6 Another dotted line shows the trajectory corresponding to a right-censored observation. The cell remained in the pre-disintegration high plateau region throughout the entire burst acquisition process, and the slow decrease in its normalized integral intensity was caused by the bleaching residual rather than disintegration.

[0051] The complete trajectory must simultaneously meet three conditions: a coefficient of determination of 0.95 or higher; a difference between the high plateau before disintegration and the low plateau after disintegration of 5 times or more the standard deviation of the reference noise within the fixed measurement mask; and the cell being within the field of view throughout the burst acquisition. The standard deviation of the reference noise is the standard deviation of the normalized integral intensity within the low plateau segment after disintegration. For cells that remain in the high plateau segment before disintegration during burst acquisition, the end time of burst acquisition is used as the right-censored observation. The right-censored observation and the complete trajectory are included in the constrained joint regression using the log-normal accelerated failure time likelihood. The time when cells subjected to higher exposure levels enter the disintegration process is generally delayed. If only cells that complete disintegration within the window are retained, under high exposure conditions, only a portion of cells that are less affected will be retained. The resulting exposure response slope will be systematically underestimated, forming a selection bias caused by retaining only those that have completed the process. Therefore, the right-censored observation is retained instead of being discarded. The nucleolar disintegration half-time is positive and right-skewed. After taking the logarithm, it approaches normality. Therefore, the log-normal form is selected.

[0052] The calibration of the internal control cells was performed in a single calibration experiment: the internal control cells were cultured separately under the same blocking conditions as the test samples, and the linear relationship between the nucleolar disintegration half-time and the total blocking exposure was measured to obtain the calibration intercept and the calibration exposure response slope. Subsequently, for each culture well, the median nucleolar disintegration half-time of the internal control cells in that well was taken, and the actual total blocking exposure of that well was inverted according to the following relationship. In the formula, E a The actual total blocking exposure for this culture well is expressed in millimoles per hour per liter; M is the median half-time of nucleolar disintegration in the internal control cells within this well, expressed in minutes; b r For calibrating the intercept, the unit is minutes; k r To calibrate the slope of the exposure response, the unit is minutes multiplied by liters per millimole per hour. A few cells may give outlier values ​​due to poor adhesion or shading from neighboring cells. The median is not sensitive to such outliers, so the median is used instead of the mean.

[0053] Figure 7 This diagram illustrates the inversion between the calibration of internal control cells and the actual total blocking exposure. Solid circles represent the values ​​measured by internal control cells under normal conditions, while hollow squares represent the values ​​measured under saturated salvage conditions. Both fall on the same solid line. This result is generated by deoxycytidine kinase knockout: internal control cells no longer respond to deoxycytidine salvage and therefore respond to exposure with the same relationship under both conditions. The intersection of the solid line and the vertical axis is the calibration intercept, marked with a hollow rhombus; the right-angled triangles in the diagram represent the slope of the calibration exposure response. The inversion process is described below. Figure 7 The horizontal and vertical dashed lines in the diagram represent the actual total blocking exposure of the well by drawing a horizontal line from the median of the half-time value of the nucleolus disintegration of the internal reference cells in the well and intersecting it with the calibration line. A vertical line is then drawn from the intersection point to intersect the horizontal axis. The resulting horizontal coordinate is the actual total blocking exposure of the well. Figure 7The nominal value for each well is marked with a vertical dotted line. The deviation between the actual total blocking exposure and the nominal value is caused by a combination of sample volume deviation, elution residue, and culture medium evaporation. In practice, the median difference between the actual and total blocking exposures should be within ±10% of the total blocking exposure as a criterion for controlled administration. If this range is exceeded, it indicates a systemic problem in the operation, which should be investigated and repeated, rather than being masked by data manipulation.

[0054] Before performing constrained joint regression, we first verify whether defining the total blocking exposure as the sum of products is appropriate. Using the nucleolar disintegration half-time of the conventional culture wells and calibration units as the observation, we fit an independent coefficient model containing the coefficients of the first and second blocking exposures. In the formula, T is the half-time of nucleolus disintegration, in minutes; T0 is the half-time of intrinsic nucleolus disintegration, in minutes; α1 is the first blocking exposure coefficient, and α2 is the second blocking exposure coefficient, both in minutes multiplied by liters per millimole per hour. The ratio of the first blocking exposure to the second blocking exposure in the nine gradient units varies with the duration of the second blocking. Furthermore, the first blocking duration of the validation unit differs from the other units, making the two coefficients identifiable under this design. When the 95% confidence interval of the difference between the first and second blocking exposure coefficients falls entirely within the preset equivalent interval, the unit exposure from the two blocking methods is deemed equivalent. Subsequently, the two can be combined into a total blocking exposure for constrained joint regression.

[0055] The constrained joint regression model uses the half-time of single-cell nucleolar disintegration as the observation and the culture wells as the random effect layer. It incorporates all observations from the conventional group culture wells, the saturation rescue group culture wells, and the two anchor points with a total blocking exposure of 0 into a single model with right-censored likelihood. In the formula, T ijk The time taken, in minutes, for the nucleolus disintegration of the i-th cell in the k-th culture well under salvage condition j; g j For the rescue status indicator, the wells in the routine group are assigned a value of 0, and the wells in the saturated rescue group are assigned a value of 1; E ak δ represents the actual total blocking exposure in the k-th well, in millimoles per hour per liter; T0 represents the intrinsic nucleolar disintegration half-time, i.e., the nucleolar disintegration half-time under conditions of no blocking and omission of deoxycytidine, in minutes; δ represents the salvage operation constant component, i.e., the displacement caused by the deoxycytidine operation itself, independent of the exposure amount, in minutes; α represents the artifact sensitivity, i.e., the rate at which the nucleolar disintegration half-time increases with the actual total blocking exposure in the conventional group wells; ρ represents the residual exposure response slope after salvage, i.e., the same growth rate in the saturated salvage group wells. Both α and ρ are in minutes multiplied by millimoles per hour; u k Let ε be the random effect of the k-th culture well. ijkThe values ​​are residuals, and both are in minutes. For the unblocked control wells, the intrinsic nucleolar disintegration half-time was directly observed at a point where the actual total blocking exposure was 0 and the salvage status indicator was 0. For the deoxycytidine-only control wells, the sum of the intrinsic nucleolar disintegration half-time and the salvage operation constant component was directly observed at a point where the actual total blocking exposure was 0 and the salvage status indicator was 1. Both methods allow for independent external anchoring of these two parameters, without relying on extrapolation from a sloped straight line to zero. The culture wells containing the two anchor points omitted blocking, resulting in dispersed cell prophase entry times during the observation period. The number of effective trajectories obtainable within the same observation window was far fewer than in synchronized culture wells, leading to a wider confidence interval when estimated solely based on this. Integrating these observations with those of nine gradient units into the same fit, where the anchor points determine the longitudinal position and the gradient units provide the number of trajectories, allows both the intrinsic nucleolar disintegration half-time and the salvage operation constant component to simultaneously obtain unbiased positions and sufficiently narrow confidence intervals.

[0056] The apparent remediation ratio is given by the artifact sensitivity and the slope of the residual exposure response after remediation, i.e. In the formula, η is the apparent salvage ratio, dimensionless. This quantity characterizes the proportion of the deoxycytidine salvage reduction exposure response slope, and its value is jointly determined by the nucleoside transport rate, deoxycytidine kinase activity, deoxycytidine deamination rate, and the regulatory state of ribonucleotide reductase. It is a composite indicator and should not be directly equated to the activity of any single step.

[0057] The preset equivalent interval uses the equivalent boundary value as the positive and negative boundary, and the equivalent boundary value is... In the formula, Δ is the equivalent threshold value, with units of minutes multiplied by liters per millimole per hour; T ref For reference, the nucleolus disintegration half-time is measured in minutes; E max With E min These represent the maximum and minimum total blocking exposure values ​​across multiple gradient units, expressed in millimole-hours per liter. This formula allows the measured values ​​from the saturated salvage group culture wells to drift by no more than 10% of the reference nucleolar disintegration half-hour over the entire exposure range. Dividing this allowable drift by the exposure range yields the maximum absolute value of the slope. When the reference nucleolar disintegration half-hour is 14.80 minutes and the total blocking exposure ranges from 30 to 72 millimole-hours per liter, the equivalent threshold is 0.0352 minutes multiplied by millimoles per liter per hour.

[0058] The determination of whether the decoupling between the blocking-induced component and the intrinsic nucleolar disintegration half-time is valid is based on a set of conditions. The primary condition is that the 95% confidence interval of the residual exposure response slope after remediation falls entirely within a pre-defined equivalent interval. This means that the measured values ​​in the saturated remediation group wells are independent of the actual total blocking exposure. In this case, the mean of the nucleolar disintegration half-time obtained in each gradient unit is a plateau estimate of the sum of the intrinsic nucleolar disintegration half-time and the remediation operation constant component. Subtracting the remediation operation constant component yields an independent estimate of the intrinsic nucleolar disintegration half-time, used for cross-checking with the intrinsic nucleolar disintegration half-time obtained from constrained joint regression. The second condition is that the lower limit of the 95% confidence interval for artifact sensitivity reaches three times or more of the equivalent threshold, confirming the existence of a distinguishable blocking-induced component in the conventional group wells. If this condition is not met, the decomposition loses its target; in this case, the residual exposure response slope falling into the equivalent interval after remediation is only because there was no component to be eliminated. The third condition is that the lower limit of the 95% confidence interval for the apparent salvage ratio reaches 0.9 or higher, confirming that the salvage reduces the vast majority of the exposure response rather than having only a weak effect. The fourth condition is that the 95% confidence interval of the difference between the mean value of the nucleolar disintegration half-time obtained from multiple gradient units in the saturated salvage group culture wells, minus the salvage operation constant component, and the reference nucleolar disintegration half-time falls within the equivalent interval of ±10% of the reference nucleolar disintegration half-time. This condition compares the high-precision estimate given by the synchronization pathway with the independent observations given by the unblocked control wells to check for any residual bias unrelated to the exposure amount. When all the above conditions are met, the reported intrinsic nucleolar disintegration half-time is the estimate obtained from constrained joint regression.

[0059] Figure 8 The diagram illustrates the dual-branch approach of the constrained joint regression and the exposure independence criterion. The horizontal axis represents the actual total blocking exposure, and the vertical axis represents the nucleolar disintegration half-time. The upper solid line represents the regression line for the conventional group culture wells, with the solid dots representing the measured values ​​of the 9 gradient units. The lower, nearly horizontal solid line represents the regression line for the saturated salvage group culture wells, with the hollow squares representing the corresponding measured values. The solid triangle at the horizontal axis of 0 represents the measured value of the unblocked control wells, and the hollow triangle represents the measured value of the deoxycytidine-only control wells. The difference between the two vertical axes represents the salvage operation constant component. Figure 8 The right-angled triangle in the diagram represents the artifact sensitivity, i.e., the slope of the regression line for the standard group; the vertical double arrow at the maximum value of the horizontal axis represents the blocking-induced component at that exposure level, i.e., the longitudinal distance between the two regression lines. The two dashed lines extending to the right from the horizontal axis 0 constitute the preset equivalent interval, with the slopes of their upper and lower boundaries representing the positive and negative equivalent thresholds, respectively. Figure 8The regression line in the conventional group sloped significantly upward, indicating that the blocking-induced component increased significantly with exposure. The regression line in the saturation-remediation group remained within the wedge-shaped range between the two dashed lines, indicating that its slope fell within the pre-defined equivalent interval, and the measured values ​​were independent of the exposure level. This nearly horizontal line reduced the blocking-induced component, which was originally inseparable from the intrinsic component, to a negligible level in a testable manner.

[0060] Whether the contributions of the two blockades satisfy additivity is verified by segmented salvage. Culture wells for the pre-salvage group and the post-salvage group are set up in three gradient units with the lowest, middle, and highest total blockade exposure, respectively. The first segment difference, the second segment difference, and the total difference are calculated, representing the difference in nucleolar disintegration half-time between the conventional group culture well and the pre-salvage group culture well, the post-salvage group culture well, and the saturated salvage group culture well, respectively. The additivity test metric is... In the formula, D1 is the first segment difference, D2 is the second segment difference, and D... 12 The sum of the differences is represented by R, and the additivity test value is represented by R. All three values ​​are in minutes. When additivity is true, the first segmented difference reflects the contribution of the first blockade, the second segmented difference reflects the contribution of the second blockade, and the sum of the two should be equal to the sum of the differences. All three differences are calculated with the wells in the standard group as the minuend. Each of the three salvage group wells has undergone one or two deoxycytidine applications. According to the aforementioned model, the additivity test value is theoretically equal to the negative value of the salvage operation constant component, not strictly zero. This conclusion is independent of the specific ratio of the first and second blockade exposures, and therefore takes the same value across the three tested gradient units. In practice, ±10% of the reference nucleolus disintegration time is used as the equivalent interval, the width of which is sufficient to accommodate the constant shift. For more stringent verification, the salvage operation constant component can be added to the additivity test value before comparison, so that the theoretical expectation of the test value returns to zero. This is an optional implementation method. The requirement is that the 95% confidence interval of the additivity test quantity falls within the equivalent interval on all three gradient units. Additivity is a requirement for the entire exposure range, and the validity of a single point does not rule out the possibility of interaction at other exposure levels. Therefore, the test is conducted separately at the low, medium, and high levels.

[0061] Figure 9 This illustrates the principle of the additivity test for segmented salvage. In the figure, the horizontal axis represents the total blocking exposure of the three tested gradient units, and the vertical axis represents the nucleolus disintegration half-time. At each exposure level, the measured values ​​of the culture wells of the conventional group, the pre-segment salvage group, the post-segment salvage group, and the saturation salvage group are marked with solid circles, hollow upper triangles, solid lower triangles, and hollow squares, respectively. The four measured values ​​at the same exposure level are connected by vertical dotted lines. Figure 9The upper left corner shows the labeling of the four culture wells in a one-to-one correspondence between the label and the name; in the figure, a difference is shown by a vertical line with an end cap at each of the three tested gradient units. From left to right, these are the first segment difference, the second segment difference, and the total difference. All three are calculated in the same way at each gradient unit. Figure 9 Two results were presented: as the exposure level increased, the distance between the culture wells of the conventional group and the culture wells of the saturated rescue group increased significantly, while the measured value of the culture wells of the saturated rescue group only increased slightly; at the unit with the highest total blocking exposure level, the first blocking exposure level and the second blocking exposure level were exactly equal, and the measured values ​​of the culture wells of the first rescue group and the culture wells of the second rescue group overlapped. This overlap constituted an independent confirmation of additivity and the definition of exposure level. Figure 9 The additivity test value marked below is -0.40 minutes for all three tested units, which is consistent with the negative value of the remedial operation constant component and falls within the equivalent range of ±1.48 minutes.

[0062] A set of exemplary data is provided to illustrate the actual results of the above procedure. The results of a patient-derived tumor cell culture are as follows: reference nucleolar disintegration half-time 14.80 minutes; deoxycytidine control well 15.20 minutes, from which the salvage operation constant component is calculated to be 0.40 minutes; intrinsic nucleolar disintegration half-time obtained from constraint combined regression is 14.80 minutes; the artifact sensitivity of the conventional group culture wells is 0.145 minutes multiplied by liters per millimole per hour, with a 95% confidence interval of 0.128 to 0.162, the lower limit of which is more than three times the equivalence threshold, i.e., 0.106; the residual exposure response slope after salvage of the saturated salvage group culture wells is 0.006 minutes multiplied by liters per millimole per hour, with a 95% confidence interval of -0.010. The value was 0.022, falling within the preset equivalent range of -0.0352 to +0.0352. The apparent salvage ratio was 0.959, with a 95% confidence interval of 0.915 to 0.986, the lower limit of which was higher than 0.9. The mean nucleolar disintegration half-time obtained from the saturated salvage group culture wells in the 9 gradient units was 15.50 minutes, which, after subtracting the salvage operation constant component, was 15.10 minutes. The difference between this value and the intrinsic nucleolar disintegration half-time obtained from the constraint joint regression was 0.30 minutes, which was equal to the product of the residual exposure response slope after salvage and the mean total blocking exposure of 49.5 mmol / L in the 9 gradient units, falling within the equivalent range of ±1.48 minutes. All the above conditions were met, therefore, it was determined that the blocking induction component and the intrinsic nucleolar disintegration half-time were decoupled, and the intrinsic nucleolar disintegration half-time was reported as 14.80 minutes.

[0063] The measured values ​​of the same sample in the conventional group culture wells increased from 19.15 minutes to 25.24 minutes as the total blocking exposure increased from 30 mmol / L to 72 mmol / L, with a coefficient of variation of 9.8% for the values ​​measured in the nine gradient units. After subtracting the blocking-induced component from the data of the same batch according to the procedure of this invention, the coefficient of variation of the intrinsic nucleolar disintegration half-time obtained from the nine gradient units decreased to 1.4%. Repeating the entire procedure on three independent batches, the inter-batch coefficient of variation of the nucleolar disintegration half-time measured under a single blocking condition was 8.3%, while the inter-batch coefficient of variation of the intrinsic nucleolar disintegration half-time output according to this invention was 2.1%. This comparison shows that the dispersion across blocking conditions and across batches mainly comes from the blocking-induced component, and the amount remaining after separating this component has stability for use as a quantitative indicator.

[0064] The parameter output provides a set of nucleolar dynamic evolution parameters, including intrinsic nucleolar disintegration half-time, artifact sensitivity, apparent salvage ratio, salvage operation constant component, mitotic entry rate, right censoring observation ratio, cell death rate, and the coefficient of variation of intrinsic nucleolar disintegration half-time obtained from three independent batches of the same test cells. The three main parameters each have independent meanings: intrinsic nucleolar disintegration half-time describes the disintegration dynamics of the nucleolar in promitosis; artifact sensitivity characterizes the intensity of the nucleolar response to replication stress, with a higher value indicating greater sensitivity to nucleotide pool imbalance; and the apparent salvage ratio characterizes the apparent efficiency of deoxycytidine replenishing the nucleotide pool along the salvage pathway, which is related to the patency of the nucleotide salvage pathway in the sample. All three parameters are given simultaneously from the same measurement and are not interchangeable.

[0065] Several optional implementation methods are given below. Nucleolar labeling is not limited to nucleolar fibroin; fluorescent protein coding sequences can also be knocked into the intrinsic sites of nucleolar phosphoproteins to label nucleolar particle components. In this case, the measured nucleolar disintegration half-time reflects the disintegration process of the particle components, and its value differs from that when labeled with nucleolar fibroin. Therefore, the labeling targets should be fixed in the same study and should not be mixed. The type of fluorescent protein can be changed according to the channel configuration of the imaging device, but it must be ensured that the nucleolar fluorescent labels of the test cells and internal control cells are spectrally distinguishable and that cross-coloring can be eliminated by linear unmixing.

[0066] Alternative approaches exist for constructing internal control cells. Besides knocking out the deoxycytidine kinase gene, deoxycytidine uptake can be blocked by knocking out the nucleotide transporter gene or by continuous treatment with its inhibitor, similarly achieving an epigenetic salvage ratio in the equivalent zero range for internal control cells. The reason for choosing to knock out deoxycytidine kinase is that this enzyme is located at the first phosphorylation step in the salvage pathway and has well-defined substrate specificity. Knockout does not affect the deoxythymidine triphosphate production pathway, thus not interfering with the response of internal control cells to exposure levels.

[0067] The number of gradient levels and their ranges can be adjusted according to sample size and throughput. When the number of available cells is limited, the number of gradient units can be reduced from 9 to 6, i.e., 2 levels for concentration and 3 levels for the second blocking duration. However, the span of the total blocking exposure should not be less than 2 times; otherwise, the confidence interval of the exposure response slope will be too wide and will be difficult to meet the lower limit condition for artifact sensitivity. The width of the equivalent interval can also be adjusted according to the application: it can be widened to 15% of the reference nucleolar disintegration half-time when used for screening, and tightened to 5% when used for quantitative comparison. The trade-off for tightening is that more replicates are needed to reduce the width of the confidence interval.

[0068] In addition to using the standard deviation of chromatin channel grayscale, the determination of entering prophase can also be based on the shrinkage rate of the nuclear region area or the decrease in the nucleocytoplasmic fluorescence intensity ratio. When using the latter, the nuclear localization fluorescent protein must be labeled separately, which will occupy an additional channel. The burst acquisition duration can be extended according to the distribution of nucleolar disintegration half-time obtained from preliminary experiments. For example, when the proportion of right censoring observations exceeds 20% within a 90-minute window, the window can be extended to 120 minutes, while the acquisition interval can be widened to 3 minutes to maintain a constant cumulative irradiance.

[0069] The constrained joint regression can be solved using the restricted maximum likelihood method, with right-censored observations contributing to the survival function based on the log-normal accelerated failure time likelihood. The 95% confidence intervals for each parameter can be given using the asymptotic method or a stratified bootstrap method with culture wells as resampling units, after 2000 resampling cycles. The latter is more robust when the number of replicates is small. Furthermore, leave-one-gradient cross-validation can be added: one gradient unit is left out sequentially, and the parameters fitted to the remaining units are used to predict the measured values ​​of the left-out units. The normalized root mean square error of the predicted values ​​should be within 10%, used to verify the applicability of the linear form across the entire exposure range.

[0070] In parallel quality control, thymidine analogs can be replaced with other clickable chemolabeled nucleoside analogs; in addition to liquid chromatography-tandem mass spectrometry, nucleotide pool determination can also be performed using polymerase-based nucleotide quantification methods, provided that deoxycytidine triphosphate and deoxythymidine triphosphate are measured simultaneously in the same sample to eliminate the influence of sample size differences on the comparison value.

[0071] The method of this invention is implemented under in vitro conditions, and the output is a set of nucleolar dynamic evolution parameters, i.e., a set of quantified characteristic parameters, which can be used in subsequent pathological analysis. The above embodiments are only used to illustrate the technical solution of this invention. Equivalent substitutions and improvements made by those skilled in the art without departing from the concept of this invention should be included within the scope of protection of this invention.

Claims

1. A method for analyzing the dynamic evolution of tumor cell nucleoli based on thymine blockade synchronization, characterized in that, Includes the following steps: Step 1: The test cells and internal control cells taken from the biopsy sample that has been excised are co-cultured in multiple culture wells. The internal control cells are tumor cell lines that have been knocked out of the deoxycytidine kinase gene and whose nucleolar fluorescent labeling is distinguishable from the spectrum of the test cells. Step 2: Apply the first blockade, intermediate release phase, second blockade, and final release phase sequentially to multiple culture wells. The first and second blockades are both performed with thymidine deoxyriboside. The multiple culture wells constitute multiple gradient units with different total blockade exposures. Each gradient unit includes a conventional group culture well and a saturated rescue group culture well. The conventional group culture wells use conventional culture medium during the intermediate and final release phases. The saturated rescue group culture wells are treated with a saturated rescue concentration of deoxycytidine during the intermediate and final release phases. Additionally, there are no blockade control wells (without thymidine deoxyriboside) and deoxycytidine control wells. Step 3: Acquire multi-channel time-series images for multiple culture wells, and measure the nucleolar disintegration half-time of the test cells and internal control cells respectively. The actual total blocking exposure of each culture well is then inverted from the nucleolar disintegration half-time of the internal control cells. The nucleolar disintegration half-time obtained from the unblocked control well is recorded as the reference nucleolar disintegration half-time. Step 4: Using the actual total blocking exposure as the independent variable, and the unblocked control well and the deoxycytidine control well as the anchor point with a total blocking exposure of 0, perform constrained joint regression on the nucleolar disintegration half-time of the conventional group culture well and the saturated salvage group culture well to obtain the intrinsic nucleolar disintegration half-time, artifact sensitivity, salvage operation constant component and salvage residual exposure response slope. When the 95% confidence interval of the salvage residual exposure response slope falls entirely into the preset equivalent interval, it is determined that the blocking induction component decoupling is established. Step 5: Output a set of nucleolar dynamic evolution parameters, including intrinsic nucleolar disintegration half-time, artifact sensitivity, and apparent salvage ratio.

2. The method according to claim 1, characterized in that: In step 2, the first blocking exposure is the product of the thymidine deoxyribonucleoside concentration and the first blocking duration; the second blocking exposure is the product of the thymidine deoxyribonucleoside concentration and the second blocking duration; and the total blocking exposure is the sum of the first and second blocking exposures. The thymidine deoxyribonucleoside concentration is the same in both the first and second blocking. Three levels of thymidine deoxyribonucleoside concentrations (1.0, 1.5, and 2.0 mmol / L) are used. The first blocking duration is 18 hours, with an intermediate release period of 9 hours. Three levels of second blocking durations (12, 15, and 18 hours) are used. The combination of thymidine deoxyribonucleoside concentration and second blocking duration constitutes nine gradient units, with the total blocking exposure ranging from 30 to 72 mmol / L. A validation unit is also included in step 2. The first blocking duration of the validation unit differs from the first blocking duration of the nine gradient units, and the total blocking exposure of the validation unit is the same as that of one of the gradient units. Three independent replicates are set up for each treatment condition.

3. The method according to claim 2, characterized in that: Before the constrained joint regression in step 4, the nucleolar disintegration half-time of the conventional group culture wells and the calibration unit is used as the observation. An independent coefficient model containing the first blocking exposure coefficient and the second blocking exposure coefficient is fitted. When the 95% confidence interval of the difference between the two coefficients falls into the preset equivalent interval, the first blocking exposure and the second blocking exposure are combined into a total blocking exposure for participation in the constrained joint regression. When the gradient unit with the same blocking exposure as the calibration unit falls into the equivalent interval of ±5% of the reference nucleolar disintegration half-time of the difference obtained from the conventional group culture wells in the 95% confidence interval of the difference between the nucleolar disintegration half-times, the definition of the total blocking exposure is deemed appropriate.

4. The method according to claim 1, characterized in that: In step 2, pre-rescue group culture wells and post-rescue group culture wells are set up in the three gradient units with the lowest, middle and highest total blocking exposure. The pre-rescue group culture wells are only given a saturated rescue concentration of deoxycytidine during the intermediate release phase, and the post-rescue group culture wells are only given a saturated rescue concentration of deoxycytidine during the final release phase. Step 4 also includes calculating the first segment difference, the second segment difference and the total difference in the three gradient units respectively. The first segment difference, the second segment difference and the total difference are the differences in nucleolar disintegration half-time obtained by the conventional group culture well and the pre-rescue group culture well, the post-rescue group culture well and the saturated rescue group culture well, respectively. When the 95% confidence interval of the value obtained by subtracting the total difference from the sum of the first segment difference and the second segment difference falls within the equivalent interval of ±10% of the reference nucleolar disintegration half-time in all three gradient units, it is determined that the blocking induction components contributed by the first blocking and the second blocking satisfy the additivity.

5. The method according to claim 1, characterized in that: The saturation salvage concentration in step 2 was determined by the gradient unit condition of the preliminary experiment with the highest total blocking exposure. The deoxycytidine concentration in the preliminary experiment was set at 5 levels: 0, 8, 24, 48 and 96 μmol / L. The lowest deoxycytidine concentration that fell within the equivalent range of ±5% of the half-time difference of nucleolar disintegration obtained from the difference of two adjacent deoxycytidine concentrations was taken as the saturation salvage concentration. The saturation salvage concentration was used uniformly for all culture wells in step 2 where deoxycytidine was applied.

6. The method according to claim 1, characterized in that: In step 1, the test cells were obtained from ex vivo biopsy samples through enzymatic digestion, primary culture, and tumor origin identification. An enhanced green fluorescent protein (GFP) coding sequence was knocked into the nucleolar fibroin intrinsic site using homologous recombination. The internal control cells had a monomeric red fluorescent protein (RFP) coding sequence knocked into the nucleolar fibroin intrinsic site. The internal control cells and test cells were co-cultured at a seeding density of 1:

9. The 95% confidence interval of the difference between the nucleolar disintegration half-time obtained from co-cultured wells and individually cultured wells fell within the equivalent interval of ±5% of the reference nucleolar disintegration half-time. The calibration intercept and calibration exposure response slope of the internal control cells were given by a single calibration experiment. In step 3, the actual total blocking exposure was retrieved by taking the difference between the median nucleolar disintegration half-time of the internal control cells in the culture wells and the calibration intercept, and dividing by the calibration exposure response slope.

7. The method according to claim 1, characterized in that: In step 3, an automated high-content live-cell imaging device was used to independently time and collect images according to the start time of the terminal release phase of each culture well. The acquisition included inspection acquisition and burst acquisition. Inspection acquisition, from 6 to 16 hours after the start of the terminal release phase, only collected images of chromatin channels labeled with chromatin fluorescent dye at 10-minute intervals. When the standard deviation of grayscale in the nucleus region reached 1.5 times or more of the average value obtained from the previous 6 rounds of inspection acquisition for the same cell, the cell was determined to have entered the prophase, and the corresponding acquisition round was recorded as the prophase determination round, and the corresponding field of view was switched to burst acquisition. Burst acquisition lasted for 90 minutes at 2-minute intervals, and simultaneously acquired images of the nucleolar channel of the cell under test, the nucleolar channel of the internal control cell, and the chromatin channel. The total time of a single round of 3-channel scanning was within 2 minutes, and the cumulative irradiation of a single cell was within 8 joules per square centimeter.

8. The method according to claim 7, characterized in that: The method for determining the nucleolar disintegration half-time in step 3 includes: obtaining a fixed measurement mask by thresholding and morphological dilation of the nucleolar channel image of the previous round; estimating the translation and rotation components round by round from the centroid and principal axis of the second central moment of the nucleus region in the chromatin channel, and applying the same rigid body transformation to the fixed measurement mask; The normalized integral intensity is obtained by quoting the integral intensity within the fixed measurement mask with respect to the bleaching reference. The bleaching reference is the round-by-round average of the integral intensity of cells in interphase within the same field of view in a mask of the same size. Using the previous round as the time origin, a 4-parameter logistic function is fitted to the time series of normalized integral intensity to obtain the high plateau before disintegration and the low plateau after disintegration. The half-time of nucleolus disintegration is the difference between the time origin and the moment when the normalized integral intensity reaches 50% of the amplitude between the high plateau before disintegration and the low plateau after disintegration. The complete trajectory is defined as the trajectory with a fitting determination coefficient of 0.95 or higher, the difference between the high plateau before disintegration and the low plateau after disintegration being 5 times or more of the standard deviation of the reference noise within the fixed measurement mask, and the cell being within the field of view throughout the burst acquisition. For cells that remain in the high plateau region before disintegration during burst acquisition, the end time of burst acquisition is used as the right censored observation. The right censored observation and the complete trajectory are included in the constrained joint regression of step 4 using the log-normal accelerated failure time likelihood.

9. The method according to claim 7, characterized in that: Step 2 also includes parallel quality control cultures and light and dye control wells. The parallel quality control cultures are cultured separately from multiple wells under identical treatment conditions. A portion of the parallel quality control cultures are pulse-labeled with thymidine analogs at the start of the terminal release phase. After fixation, they are subjected to dual-parameter flow cytometry to measure thymidine analog incorporation and DNA content. Synchronization is defined as 75% or higher of early S-phase cells with diploid DNA content and positive thymidine analog incorporation. Another portion of the parallel quality control cultures are subjected to liquid chromatography-tandem mass spectrometry to determine the ratio of deoxycytidine triphosphate (doxycytidine triphosphate) to deoxythymidine triphosphate (doxycytidine triphosphate) one hour after the start of the terminal release phase. The ratio of doxycytidine triphosphate (doxycytidine triphosphate) to doxythymidine triphosphate (doxycytidine triphosphate) is used as the reference for the saturation salvage group wells. The deviation of the content ratio from the content ratio of the unblocked control wells within 20% is used as the backfill criterion. The mitotic entry rate of the illumination and dye control wells is measured under three conditions: burst acquisition with chromatin fluorescent dye applied, bright-field inspection acquisition with chromatin fluorescent dye applied, and bright-field inspection acquisition without chromatin fluorescent dye. Bright-field inspection acquisition is inspection acquisition performed by replacing fluorescence excitation with transmitted illumination. The criterion for acceptable illumination and dye influence is that the 95% confidence interval of the difference between the mitotic entry rates obtained under any two conditions falls within the equivalent interval of ±3 percentage points. When the synchronization criterion, the backfill criterion, and the acceptable illumination and dye influence criterion are all met, step 3 is continued.

10. The method according to claim 1, characterized in that: In step 4, the predefined equivalent interval uses the equivalent boundary value as the positive and negative boundary. The equivalent boundary value is 10% of the reference nucleolar disintegration half-time divided by the difference between the maximum and minimum total blocking exposure values ​​in multiple gradient units. Constrained joint regression ensures that the nucleolar disintegration half-time of the conventional group culture wells is equal to the sum of the products of the intrinsic nucleolar disintegration half-time, artifact sensitivity, and actual total blocking exposure. It also ensures that the nucleolar disintegration half-time of the saturated salvage group culture wells is equal to the sum of the products of the intrinsic nucleolar disintegration half-time, the salvage operation constant component, the salvage residual exposure response slope, and the actual total blocking exposure. The salvage operation constant component is the difference between the nucleolar disintegration half-time obtained from the deoxycytidine control well and the unblocked control well. The apparent salvage ratio is the difference between 1 and the ratio of the salvage residual exposure response slope to the artifact sensitivity. The blocking induction component is the nucleolar disintegration half-time that varies with the actual total blocking exposure. The components of the exposure change were interrupted; the combined regression was constrained with the single-cell nucleolar disintegration half-time as the observation and the culture wells as the random effect layer; the conditions for determining the decoupling of the blocking-induced components also included the lower limit of the 95% confidence interval of the artifact sensitivity reaching 3 times or more of the equivalent threshold, the lower limit of the 95% confidence interval of the apparent salvage ratio reaching 0.9 or more, and the 95% confidence interval of the difference between the mean value of the nucleolar disintegration half-time obtained by the saturated salvage group culture wells in multiple gradient units minus the salvage operation constant component and the reference nucleolar disintegration half-time falling within the equivalent interval of ±10% of the reference nucleolar disintegration half-time; the set of nucleolar dynamic evolution parameters output in step 5 also included the salvage operation constant component, mitotic entry rate, right censoring observation ratio, cell death rate, and the coefficient of variation of the intrinsic nucleolar disintegration half-time among 3 independent batches.

Images