Development of a fit-for-purpose multi-marker panel for early diagnosis of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) suffers from lack of an effective diagnostic method, which hampers improvement in patient survival. Carbohydrate antigen 19-9 (CA19-9) is the only FDA-approved blood biomarker for PDAC, yet its clinical utility is limited due to suboptimal performance. Liquid chromatography-mass spectrometry (LC-MS) has emerged as a burgeoning technology in clinical proteomics for discovery, verification, and validation of novel biomarkers. A plethora of protein biomarker candidates for PDAC have been identified using LC-MS, yet few has successfully transitioned into clinical practice. This translational standstill is owed partly to insufficient considerations of practical needs and perspectives of clinical implementation during biomarker development pipelines, such as demonstrating analytical robustness of proposed biomarkers which is critical for transitioning from research-grade to clinical-grade assays. Moreover, throughput and cost-effectiveness of proposed assays ought to be considered concomitantly from early phases of the biomarker pipelines for enhancing widespread adoption in clinical settings. Here, we developed a fit-for-purpose multi-marker panel for PDAC diagnosis by consolidating analytically robust biomarkers as well as employing a relatively simple LC-MS protocol. In discovery phase, we comprehensively surveyed putative PDAC biomarkers from both in-house data and prior studies. In verification phase, we developed a multiple-reaction monitoring (MRM)-MS-based proteomic assay using surrogate peptides that passed stringent analytical validation tests. We adopted a high-throughput protocol including short gradient (<10 min) and simple sample preparation (no depletion or enrichment steps). Additionally, we developed our assay using serum samples, which are usually the preferred biospecimen in clinical settings. We developed predictive models based on our final panel of 12 protein biomarkers combined with CA19-9, which showed improved diagnostic performance compared to using CA19-9 alone in discriminating PDAC from non-PDAC controls including healthy individuals and patients with benign pancreatic diseases. A large-scale clinical validation is underway to demonstrate the clinical validity of our novel panel.

Safety of Gonadal Tissue-Derived Mesenchymal Stem Cell Therapy in Geriatric Dogs with Chronic Disease.

Ensuring the safety of mesenchymal stem cell (MSC) therapy is a fundamental requirement in clinical practice. This study aimed to assess the safety of using gonadal tissue-derived MSCs (n = 10) compared to the commonly utilized adipose tissue-derived MSCs (n = 9) in geriatric dogs with chronic diseases. All participants received allogeneic MSC therapy, and no allergic reactions due to allogeneic cell immunogenicity were noted. Both groups showed no adverse changes in physical exams or hematological parameters before and after therapy. Importantly, there were no instances of tumor formation or growth post-treatment in either group. The findings demonstrated that dogs treated with gonadal tissue-derived MSCs experienced no clinical adverse effects. However, clinical adverse effects were reported in one case of adipose tissue-derived MSC therapy. Despite limitations in monitoring beyond one year and constraints due to a small and diverse patient group, this pioneering study validates the safe use of gonadal tissue-derived MSCs in aged companion animals. It underscores the potential of utilizing tissues from neutering procedures to advance regenerative medicine and expand cell banks and therapy options for companion animals.

Case Series: Computed Tomography Features of Extraskeletal Osteosarcoma in Six Dogs.

The objective of the present case series was to investigate the various computed tomography findings of six dogs diagnosed with extraskeletal osteosarcoma (exOSA) at several locations. Among the tumors evaluated, four were subcutaneous, one was mammary, and one involved the intestinal tract. Intralesional mineralization was observed in all six dogs. Most of the tumors were moderately calcified, exhibited amorphous mineralization, and were heterogeneous on post-contrast imaging. Three of the tumors were peripherally enhanced, and regional lymphadenopathy was identified in two of the dogs, which was presumed to be metastatic. No lymph node calcification was reported. Although the presence of intralesional mineralization is not a pathognomonic finding, it was consistently identified in the present case series. Therefore, exOSA should be considered in the differential diagnosis when mineralization occurs in a mass unrelated to osseous structures.

Exploring the Tumor-Associated Risk of Mesenchymal Stem Cell Therapy in Veterinary Medicine.

Mesenchymal stem cell (MSC) therapy has been actively applied in veterinary regenerative medicine to treat various canine and feline diseases. With increasing emphasis on safe cell-based therapies, evaluations of their tumorigenic potential are in great demand. However, a direct confirmation of whether tumors originate from stem cells or host cells is not easily achievable. Additionally, previous studies evaluating injections of high doses of MSCs into nude mice did not demonstrate tumor formation. Recent research focused on optimizing MSC-based therapies for veterinary patients, such as MSC-derived extracellular vesicles in treating different diseases. This progress also signifies a broader shift towards personalized veterinary medicine, where treatments can be tailored to individual pets based on their unique genetic profiles. These findings related to different treatments using MSCs emphasize their future potential for veterinary clinical applications. In summary, because of lower tumor-associated risk of MSCs as compared to embryonic and induced pluripotent stem cells, MSCs are considered a suitable source for treating various canine and feline diseases.

Radiographic and computed tomographic characteristics of intraluminal tracheal adenoma in a cat.

A 13-year-old spayed female Persian cat presented with dyspnea and nasal discharge. Thoracic radiography revealed a dome-shaped soft-tissue opacity in the carina. Computed tomography confirmed a soft tissue-attenuating mass in the carina and the left and right proximal main bronchi that appeared to arise from the tracheal wall. Tracheoscopy revealed an intraluminal broad-based mass with multilobulated borders at the same location. Histopathological evaluation revealed a benign neoplastic process of the glandular epithelial lineage, which was considered an adenoma. Tracheal adenomas should be included in the differential diagnosis of tracheal masses.

Non-canonical pathway for Rb inactivation and external signaling coordinate cell-cycle entry without CDK4/6 activity.

Cyclin-dependent kinases 4 and 6 (CDK4/6) are critical for initiating cell proliferation by inactivating the retinoblastoma (Rb) protein. However, mammalian cells can bypass CDK4/6 for Rb inactivation. Here we show a non-canonical pathway for Rb inactivation and its interplay with external signals. We find that the non-phosphorylated Rb protein in quiescent cells is intrinsically unstable, offering an alternative mechanism for initiating E2F activity. Nevertheless, this pathway incompletely induces Rb-protein loss, resulting in minimal E2F activity. To trigger cell proliferation, upregulation of mitogenic signaling is required for stabilizing c-Myc, thereby augmenting E2F activity. Concurrently, stress signaling promotes Cip/Kip levels, competitively regulating cell proliferation with mitogenic signaling. In cancer, driver mutations elevate c-Myc levels, facilitating adaptation to CDK4/6 inhibitors. Differentiated cells, despite Rb-protein loss, maintain quiescence through the modulation of c-Myc and Cip/Kip levels. Our findings provide mechanistic insights into an alternative model of cell-cycle entry and the maintenance of quiescence.

Palmar-plantar erythrodysesthesia syndrome resulting from toceranib phosphate in a dog with apocrine gland anal sac adenocarcinoma: a case report.

An 11-year-old neutered male Miniature Poodle with a stage 3 apocrine gland adenocarcinoma was started on chemotherapy with toceranib phosphate after surgery. Beginning on day 10 of toceranib, the dog's foot pads became erythematous and hyperkeratinized. The dog complained of pain, inability to walk, depression, and loss of appetite. The symptoms resolved when toceranib was discontinued and reappeared when toceranib was resumed. Grade 3 palmar-plantar erythrodysesthesia was identified as an adverse event of toceranib based on the VCOG-CTCAE and Naranjo scale. Although very rare in veterinary medicine, clinicians should consider that palmar-plantar erythrodysesthesia can occur after toceranib administration.

Angiopoietin-2 blockade suppresses growth of liver metastases from pancreatic neuroendocrine tumors by promoting T cell recruitment.

Improving the management of metastasis in pancreatic neuroendocrine tumors (PanNETs) is critical, as nearly half of patients with PanNETs present with liver metastases, and this accounts for the majority of patient mortality. We identified angiopoietin-2 (ANGPT2) as one of the most upregulated angiogenic factors in RNA-Seq data from human PanNET liver metastases and found that higher ANGPT2 expression correlated with poor survival rates. Immunohistochemical staining revealed that ANGPT2 was localized to the endothelial cells of blood vessels in PanNET liver metastases. We observed an association between the upregulation of endothelial ANGPT2 and liver metastatic progression in both patients and transgenic mouse models of PanNETs. In human and mouse PanNET liver metastases, ANGPT2 upregulation coincided with poor T cell infiltration, indicative of an immunosuppressive tumor microenvironment. Notably, both pharmacologic inhibition and genetic deletion of ANGPT2 in PanNET mouse models slowed the growth of PanNET liver metastases. Furthermore, pharmacologic inhibition of ANGPT2 promoted T cell infiltration and activation in liver metastases, improving the survival of mice with metastatic PanNETs. These changes were accompanied by reduced plasma leakage and improved vascular integrity in metastases. Together, these findings suggest that ANGPT2 blockade may be an effective strategy for promoting T cell infiltration and immunostimulatory reprogramming to reduce the growth of liver metastases in PanNETs.

Sequential activation of E2F via Rb degradation and c-Myc drives resistance to CDK4/6 inhibitors in breast cancer.

Cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) are key therapeutic agents in the management of metastatic hormone-receptor-positive breast cancer. However, the emergence of drug resistance limits their long-term efficacy. Here, we show that breast cancer cells develop CDK4/6i resistance via a sequential two-step process of E2F activation. This process entails retinoblastoma (Rb)-protein degradation, followed by c-Myc-mediated amplification of E2F transcriptional activity. CDK4/6i treatment halts cell proliferation in an Rb-dependent manner but dramatically reduces Rb-protein levels. However, this reduction in Rb levels insufficiently induces E2F activity. To develop CDK4/6i resistance, upregulation or activating mutations in mitogenic or hormone signaling are required to stabilize c-Myc levels, thereby augmenting E2F activity. Our analysis of pre-treatment tumor samples reveals a strong correlation between c-Myc levels, rather than Rb levels, and poor therapeutic outcomes after CDK4/6i treatment. Moreover, we propose that proteasome inhibitors can potentially reverse CDK4/6i resistance by restoring Rb levels.

Machine Learning of Physiologic Waveforms and Electronic Health Record Data: A Large Perioperative Data Set of High-Fidelity Physiologic Waveforms.

Perioperative morbidity and mortality are significantly associated with both static and dynamic perioperative factors. The studies investigating static perioperative factors have been reported; however, there are a limited number of previous studies and data sets analyzing dynamic perioperative factors, including physiologic waveforms, despite its clinical importance. To fill the gap, the authors introduce a novel large size perioperative data set: Machine Learning Of physiologic waveforms and electronic health Record Data (MLORD) data set. They also provide a concise tutorial on machine learning to illustrate predictive models trained on complex and diverse structures in the MLORD data set.

Preclinical Study on Biodistribution of Mesenchymal Stem Cells after Local Transplantation into the Brain.

Therapeutic efficacy of mesenchymal stem cells (MSCs) is determined by biodistribution and engraftment in vivo. Compared to intravenous infusion, biodistribution of locally transplanted MSCs are partially understood. Here, we performed a pharmacokinetics (PK) study of MSCs after local transplantation. We grafted human MSCs into the brains of immune-compromised nude mice. Then we extracted genomic DNA from brains, lungs, and livers after transplantation over a month. Using quantitative polymerase chain reaction with human Alu-specific primers, we analyzed biodistribution of the transplanted cells. To evaluate the role of residual immune response in the brain, MSCs expressing a cytosine deaminase (MSCs/CD) were used to ablate resident immune cells at the injection site. The majority of the Alu signals mostly remained at the injection site and decreased over a week, finally becoming undetectable after one month. Negligible signals were transiently detected in the lung and liver during the first week. Suppression of Iba1-positive microglia in the vicinity of the injection site using MSCs/CD prolonged the presence of the Alu signals. After local transplantation in xenograft animal models, human MSCs remain predominantly near the injection site for limited time without disseminating to other organs. Transplantation of human MSCs can locally elicit an immune response in immune compromised animals, and suppressing resident immune cells can prolong the presence of transplanted cells. Our study provides valuable insights into the in vivo fate of locally transplanted stem cells and a local delivery is effective to achieve desired dosages for neurological diseases.

THOC2 expression and its impact on 5-fluorouracil resistance in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with poor prognosis and limited treatment options. While 5-fluorouracil (5-FU) has not been widely employed in GBM therapy, emerging research indicates its potential for effectiveness when combined with advanced drug delivery systems to improve its transport to brain tumors. This study aims to investigate the role of THOC2 expression in 5-FU resistance in GBM cell lines. We evaluated diverse GBM cell lines and primary glioma cells for 5-FU sensitivity, cell doubling times, and gene expression. We observed a significant correlation between THOC2 expression and 5-FU resistance. To further investigate this correlation, we selected five GBM cell lines and developed 5-FU resistant GBM cells, including T98FR cells, through long-term 5-FU treatment. In 5-FU challenged cells, THOC2 expression was upregulated, with the highest increase in T98FR cells. THOC2 knockdown in T98FR cells reduced 5-FU IC50 values, confirming its role in 5-FU resistance. In a mouse xenograft model, THOC2 knockdown attenuated tumor growth and extended survival duration after 5-FU treatment. RNA sequencing identified differentially expressed genes and alternative splicing variants in T98FR/shTHOC2 cells. THOC2 knockdown altered Bcl-x splicing, increasing pro-apoptotic Bcl-xS expression, and impaired cell adhesion and migration by reducing L1CAM expression. These results suggest that THOC2 plays a crucial role in 5-FU resistance in GBM and that targeting THOC2 expression could be a potential therapeutic strategy for improving the efficacy of 5-FU-based combination therapies in GBM patients.

Enhanced antitumor efficacy of mesenchymal stem cells expressing cytosine deaminase and 5-fluorocytosine combined with α-galactosylceramide in a colon cancer model.

Cancer immunotherapy has emerged as a promising approach for treating various malignancies. In this study, we investigated the combined therapeutic effects of mesenchymal stem cells expressing cytosine deaminase (MSC/CD) and 5-fluorocytosine (5-FC) with α-galactosylceramide (α-GalCer) in a colon cancer model. Our findings demonstrated that the combination of MSC/CD, 5-FC, and α-GalCer resulted in enhanced antitumor activity compared to the individual treatments. This was evidenced by increased infiltration of immune cells, such as natural killer T (NKT) cells, antigen-presenting cells (APCs), T cells, and natural killer (NK) cells, in the tumor microenvironment, as well as elevated expression of proinflammatory cytokines and chemokines. Furthermore, we observed no significant hepatotoxicity following the combined treatment. Our study highlights the potential therapeutic benefits of combining MSC/CD, 5-FC, and α-GalCer for colon cancer treatment and contributes valuable insights to the field of cancer immunotherapy. Future research should focus on elucidating the underlying mechanisms and exploring the applicability of these findings to other cancer types and immunotherapy strategies.

Measurement of brainstem diameter in small-breed dogs using magnetic resonance imaging.

Measurement of brainstem diameters (midbrain, pons, and medulla oblongata)is of potential clinical significance, as changes in brainstem size may decrease or increase due to age, neurodegenerative disorders, or neoplasms. In human medicine, numerous studies have reported the normal reference range of brainstem size, which is hitherto unexplored in veterinary medicine, particularly for small-breed dogs. Therefore, this study aims to investigate the reference range of brainstem diameters in small-breed dogs and to correlate the measurements with age, body weight (BW), and body condition score (BCS). Herein, magnetic resonance (MR) images of 544 small-breed dogs were evaluated. Based on the exclusion criteria, 193 dogs were included in the midbrain and pons evaluation, and of these, 119 dogs were included in the medulla oblongata evaluation. Using MR images, the height and width of the midbrain, pons, and medulla oblongata were measured on the median and transverse plane on the T1-weighted image. For the medulla oblongata, two points were measured for each height and width. The mean values of midbrain height (MH), midbrain width (MW), pons height (PH), pons width (PW), medulla oblongata height at the fourth ventricle level (MOHV), medulla oblongata height at the cervicomedullary (CM) junction level (MOHC), rostral medulla oblongata width (RMOW), and caudal medulla oblongata width (CMOW) were 7.18 ± 0.56 mm, 17.42 ± 1.21 mm, 9.73 ± 0.64 mm, 17.23 ± 1.21 mm, 6.06 ± 0.53 mm, 5.77 ± 0.40 mm, 18.93 ± 1.25 mm, and 10.12 ± 1.08 mm, respectively. No significant differences were found between male and female dogs for all the measurements. A negative correlation was found between age and midbrain diameter, including MH (p < 0.001) and MW (p = 0.002). All brainstem diameters were correlated positively with BW (p < 0.05). No significant correlation was found between BCS and all brainstem diameters. Brainstem diameters differed significantly between breeds (p < 0.05), except for MW (p = 0.137). This study assessed linear measurements of the brainstem diameter in small-breed dogs. We suggest that these results could be useful in assessing abnormal conditions of the brainstem in small-breed dogs.

Assessment of Risks and Benefits of Using Antibiotics Resistance Genes in Mesenchymal Stem Cell-Based Ex-Vivo Therapy.

Recently, ex-vivo gene therapy has emerged as a promising approach to enhance the therapeutic potential of mesenchymal stem cells (MSCs) by introducing functional genes in vitro. Here, we explored the need of using selection markers to increase the gene delivery efficiency and evaluated the potential risks associated with their use in the manufacturing process. We used MSCs/CD that carry the cytosine deaminase gene (CD) as a therapeutic gene and a puromycin resistance gene (PuroR) as a selection marker. We evaluated the correlation between the therapeutic efficacy and the purity of therapeutic MSCs/CD by examining their anti-cancer effect on co-cultured U87/GFP cells. To simulate in vivo horizontal transfer of the PuroR gene in vivo, we generated a puromycin-resistant E. coli (E. coli/PuroR) by introducing the PuroR gene and assessed its responsiveness to various antibiotics. We found that the anti-cancer effect of MSCs/CD was directly proportional to their purity, suggesting the crucial role of the PuroR gene in eliminating impure unmodified MSCs and enhancing the purity of MSCs/CD during the manufacturing process. Additionally, we found that clinically available antibiotics were effective in inhibiting the growth of hypothetical microorganism, E. coli/PuroR. In summary, our study highlights the potential benefits of using the PuroR gene as a selection marker to enhance the purity and efficacy of therapeutic cells in MSC-based gene therapy. Furthermore, our study suggests that the potential risk of horizontal transfer of antibiotics resistance genes in vivo can be effectively managed by clinically available antibiotics.

Quantifiable peptide library bridges the gap for proteomics based biomarker discovery and validation on breast cancer.

Mass spectrometry (MS) based proteomics is widely used for biomarker discovery. However, often, most biomarker candidates from discovery are discarded during the validation processes. Such discrepancies between biomarker discovery and validation are caused by several factors, mainly due to the differences in analytical methodology and experimental conditions. Here, we generated a peptide library which allows discovery of biomarkers in the equal settings as the validation process, thereby making the transition from discovery to validation more robust and efficient. The peptide library initiated with a list of 3393 proteins detectable in the blood from public databases. For each protein, surrogate peptides favorable for detection in mass spectrometry was selected and synthesized. A total of 4683 synthesized peptides were spiked into neat serum and plasma samples to check their quantifiability in a 10 min liquid chromatography-MS/MS run time. This led to the PepQuant library, which is composed of 852 quantifiable peptides that cover 452 human blood proteins. Using the PepQuant library, we discovered 30 candidate biomarkers for breast cancer. Among the 30 candidates, nine biomarkers, FN1, VWF, PRG4, MMP9, CLU, PRDX6, PPBP, APOC1, and CHL1 were validated. By combining the quantification values of these markers, we generated a machine learning model predicting breast cancer, showing an average area under the curve of 0.9105 for the receiver operating characteristic curve.

Kinetics of RTK activation determine ERK reactivation and resistance to dual BRAF/MEK inhibition in melanoma.

The combination of BRAF and MEK inhibitors (BRAFi/MEKi) has shown promising response rates in treating BRAF-mutant melanoma by inhibiting ERK activation. However, treatment efficacy is limited by the emergence of drug-tolerant persister cells (persisters). Here, we show that the magnitude and duration of receptor tyrosine kinase (RTK) activation determine ERK reactivation and persister development. Our single-cell analysis reveals that only a small subset of melanoma cells exhibits effective RTK and ERK activation and develops persisters, despite uniform external stimuli. The kinetics of RTK activation directly influence ERK signaling dynamics and persister development. These initially rare persisters form major resistant clones through effective RTK-mediated ERK activation. Consequently, limiting RTK signaling suppresses ERK activation and cell proliferation in drug-resistant cells. Our findings provide non-genetic mechanistic insights into the role of heterogeneity in RTK activation kinetics in ERK reactivation and BRAFi/MEKi resistance, suggesting potential strategies for overcoming drug resistance in BRAF-mutant melanoma.

Angiopoietin-2-Dependent Spatial Vascular Destabilization Promotes T-cell Exclusion and Limits Immunotherapy in Melanoma.

T-cell position in the tumor microenvironment determines the probability of target encounter and tumor killing. CD8+ T-cell exclusion from the tumor parenchyma is associated with poor response to immunotherapy, and yet the biology that underpins this distinct pattern remains unclear. Here we show that the vascular destabilizing factor angiopoietin-2 (ANGPT2) causes compromised vascular integrity in the tumor periphery, leading to impaired T-cell infiltration to the tumor core. The spatial regulation of ANGPT2 in whole tumor cross-sections was analyzed in conjunction with T-cell distribution, vascular integrity, and response to immunotherapy in syngeneic murine melanoma models. T-cell exclusion was associated with ANGPT2 upregulation and elevated vascular leakage at the periphery of human and murine melanomas. Both pharmacologic and genetic blockade of ANGPT2 promoted CD8+ T-cell infiltration into the tumor core, exerting antitumor effects. Importantly, the reversal of T-cell exclusion following ANGPT2 blockade not only enhanced response to anti-PD-1 immune checkpoint blockade therapy in immunogenic, therapy-responsive mouse melanomas, but it also rendered nonresponsive tumors susceptible to immunotherapy. Therapeutic response after ANGPT2 blockade, driven by improved CD8+ T-cell infiltration to the tumor core, coincided with spatial TIE2 signaling activation and increased vascular integrity at the tumor periphery where endothelial expression of adhesion molecules was reduced. These data highlight ANGPT2/TIE2 signaling as a key mediator of T-cell exclusion and a promising target to potentiate immune checkpoint blockade efficacy in melanoma. SIGNIFICANCE: ANGPT2 limits the efficacy of immunotherapy by inducing vascular destabilization at the tumor periphery to promote T-cell exclusion.

The usefulness of a three-protein signature blood assay (Mastocheck®) for follow-up after breast cancer surgery.

PURPOSE: Mastocheck®, a proteomic-based blood assay, has been developed for early diagnosis of breast cancer. The purpose of this study is whether Mastocheck® is useful as a postoperative follow-up. METHODS: A total of 255 patients were analyzed. The patients were classified into longitudinal monitoring and recurrence/nonrecurrence cohorts. The longitudinal monitoring cohort consisted of 111 patients. In this cohort, blood analyses were performed three times (before surgery, 8 weeks after surgery, and between 6 months and one year after surgery), and a comparative analysis of the values of Mastocheck® and individual proteins at each time point was performed. The recurrence/nonrecurrence cohort consisted of 144 patients who had been followed up for more than 1 year, and the blood marker values at the time of local recurrence were compared to those of nonrecurrence patients. RESULTS: In the longitudinal monitoring cohort analysis, in 81 of 111 patients were diagnosed with breast cancer with Mastocheck® and the sensitivity was 73.0%. Of 111 patients in the longitudinal monitoring cohort, 108 had two blood analyses (before and 8 weeks after surgery), and three serial blood analyses were performed on 53 patients. The Mastocheck® value that were in the cancer range of 73.0% (in 81 of 111 patients) of patients before surgery, was within the normal range of 68.5% (in 74 of 108 patients) at 8 weeks after surgery and 88.7% (in 47 of 53 patients) from 6 months to 1 year after surgery. The value of Mastocheck® was significantly decreased after surgery compared to before surgery (p < 0.001). In the recurrence/nonrecurrence cohort analysis, the Mastocheck® values were in the cancer range in 38 out of 63 recurrence patients and within the normal range in 66 of 81 nonrecurrence patients (sensitivity of 60.3% and specificity of 80.2%). CONCLUSIONS: Mastocheck® is expected to be used as a blood marker tool to aid in the early detection of recurrence during follow-up after breast cancer surgery.

CDK4/6 initiates Rb inactivation and CDK2 activity coordinates cell-cycle commitment and G1/S transition.

External signaling controls cell-cycle entry until cells irreversibly commit to the cell cycle to ensure faithful DNA replication. This process is tightly regulated by cyclin-dependent kinases (CDKs) and the retinoblastoma protein (Rb). Here, using live-cell sensors for CDK4/6 and CDK2 activities, we propose that CDK4/6 initiates Rb inactivation and CDK2 activation, which coordinates the timing of cell-cycle commitment and sequential G1/S transition. Our data show that CDK4/6 activation induces Rb inactivation and thereby E2F activation, driving a gradual increase in CDK2 activity. We found that rapid CDK4/6 inhibition can reverse cell-cycle entry until CDK2 activity reaches to high levels. This suggests that high CDK2 activity is required to initiate CDK2-Rb positive feedback and CDK4/6-indpendent cell-cycle progression. Since CDK2 activation also facilitates initiation of DNA replication, the timing of CDK2-Rb positive feedback is coupled with the G1/S transition. Our experiments, which acutely increased CDK2 activity by cyclin E1 overexpression, indicate that cells commit to the cell cycle before triggering DNA replication. Together, our data suggest that CDK4/6 inactivates Rb to begin E2F and CDK2 activation, and high CDK2 activity is necessary and sufficient to generate a bistable switch for Rb phosphorylation before DNA replication. These findings highlight how cells initiate the cell cycle and subsequently commit to the cell cycle before the G1/S transition.