Cancer Patient-derived Xenograft Mouse Research Articles

NIR light triggered intracellular polymerization via nanoparticles containing acrylates prodrugs and azo-polymers for inhibiting cisplatin efflux for combined chemotherapy and immunotherapy

Despite the high clinical success of cisplatin, this compound is associated with insufficient intracellular uptake and quick excretion from cancer cells, resulting in poor drug efficacy. To overcome these limitations, herein, a nanoparticle formulation for efficient drug delivery and high therapeutic effect towards challenging cancer tumors by multimodal chemotherapy and immunotherapy is reported. The nanoparticles are generated upon self-assembly of a polymerizable Pt(IV) prodrug with axial acrylates, a near-infrared II polymer that can generate heat, and a thermosensitive polymer with azo bonds that can generate radicals upon exposure to heat. The nanoparticles are found with a higher cellular uptake than the molecular drug cisplatin. Upon exposure to irradiation at 1064 nm, the azo bonds are broken, and therapeutically active radicals as well as the Pt(IV) complex simultaneously released. Due to the presence of reactive acrylate moieties, the metal complex is able to undergo in-situ polymerization inside the cancer cells, resulting in the formation of a cross-linked polymeric network and therefore the retention of the cisplatin and reduction of the cisplatin efflux in the cancer cells, triggering cell death by a combination of apoptosis and immunogenic cell death. Based on these promising effects, the therapeutic efficiency is evaluated in a clinically highly challenging ovarian cancer patient-derived xenograft mouse model as well as an ID8 subcutaneous tumor-bearing C57BL/6 mouse model. This study reports on the first example of a nanoparticle delivery system with Pt(IV) prodrugs that can polymerize within cancer cells, thereby resulting in a decrease in cisplatin efflux, efficient DNA damage, and effective immunogenic response.

Abstract 1625: c-Myc inhibition in prostate cancer cell lines

Abstract The proto-oncogene c-Myc is a transcription factor that is upregulated in several human cancers. Despite its key role, therapeutic targeting of c-Myc remains a challenge because of a disordered protein tertiary structure. The basic helical structure and zipper protein of c-Myc forms an obligate heterodimer with its partner MAX to function as a transcription factor. An attractive strategy is to inhibit MYC/MAX dimerization to decrease c-Myc transcriptional function. Several methods have been described to inhibit MYC/MAX dimerization including small molecular inhibitors and proteomimetics. We used NOD SCID gamma mice (NGS mice) that are immunodeficient, non-obese diabetic laboratory mice (Jackson Laboratories, Cat Number J000077451) carrying patient-derived xenograft (PDX) following implantation of grade IV adenocarcinoma of the prostate from a patient into the flanks of each mouse. For maximum tolerable dose studies, we used C57BL/6 wild type mice (Jackson Laboratories, Cat Number J000664). We performed a maximum dose tolerability study with the drug 3JC48-3 up to 100 mg/kg to determine limiting toxicity. We monitored the rate of growth for 2 weeks and measured the final volume of the tumors. Immunoprecipitation studies were performed using the prostate cancer cell line, PC-3, that were treated with the drug 3JC48-3 at different doses (0, 5, 10, and 20uM) for 24 hours and subsequently lysed. We found 3JC48-3 decreases prostate cancer growth and viability in a dose-dependent fashion in vitro. We confirmed the inhibition of MYC/MAX dimerization by 3JC48-3 using immunoprecipitation experiments. Normal mice and mice with patient-derived prostate cancer xenografts (PDX) tolerated intraperitoneal injections of 3JC48-3 up to 100 mg/kg body weight without dose-limiting toxicity. Preliminary results in these PDX mouse models suggest that 3JC48-3 may be effective in decreasing the rate of tumor growth. Our study demonstrates that 3JC48-3 is a potent MYC/MAX heterodimerization inhibitor that decreases prostate cancer growth and viability in association with upregulation of both PrKD1 expression and kinase activity. In addition, 3JC48-3 is well tolerated in prostate cancer PDX mice models and may be a candidate drug for further preclinical development. Citation Format: Carlos Riveros, Victor Chalfant, Sanjeev Shukla, Teruko Osumi, KC Balaji. c-Myc inhibition in prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1625.

3JC48-3: Novel MYC inhibition in advanced prostate cancer.

181 Background: The proto-oncogene c-Myc is a transcription factor that is upregulated in several human cancers. Despite its key role, therapeutic targeting of c-Myc remains a challenge because of a disordered protein tertiary structure. The basic helical structure and zipper protein of c-Myc forms an obligate heterodimer with its partner MAX to function as a transcription factor. An attractive strategy is to inhibit MYC/MAX dimerization to decrease c-Myc transcriptional function. Several methods have been described to inhibit MYC/MAX dimerization including small molecular inhibitors and proteomimetics. Methods: We used NOD SCID gamma mice (NGS mice) that are immunodeficient, non-obese diabetic laboratory mice (Jackson Laboratories, Cat #J000077451) carrying patient-derived xenograft (PDX) following implantation of grade IV adenocarcinoma of the prostate from a patient into the flanks of each mouse. For maximum tolerable dose studies, we used C57BL/6 wild type mice (Jackson Laboratories, Cat #J000664). We performed a maximum dose tolerability study with the drug 3JC48-3 up to 100 mg/kg to determine limiting toxicity. We monitored the rate of growth for 2 weeks and measured the final volume of the tumors. Immunoprecipitation studies were performed using the prostate cancer cell line, PC-3, that were treated with the drug 3JC48-3 at different doses (0, 5, 10, and 20uM) for 24 hours and subsequently lysed. Results: We found 3JC48-3 decreases prostate cancer growth and viability in a dose-dependent fashion in vitro. We confirmed the inhibition of MYC/MAX dimerization by 3JC48-3 using immunoprecipitation experiments. Normal mice and mice with patient-derived prostate cancer xenografts (PDX) tolerated intraperitoneal injections of 3JC48-3 up to 100 mg/kg body weight without dose-limiting toxicity. Preliminary results in these PDX mouse models suggest that 3JC48-3 may be effective in decreasing the rate of tumor growth. Conclusions: Our study demonstrates that 3JC48-3 is a potent MYC/MAX heterodimerization inhibitor that decreases prostate cancer growth and viability in association with upregulation of both PrKD1 expression and kinase activity. In addition, 3JC48-3 is well tolerated in prostate cancer PDX mice models and may be a candidate drug for further preclinical development.

Targeting AKR1B1 inhibits glutathione de novo synthesis to overcome acquired resistance to EGFR-targeted therapy in lung cancer.

Acquired resistance represents a bottleneck to molecularly targeted therapies such as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment in lung cancer. A deeper understanding of resistance mechanisms can provide insights into this phenomenon and help to develop additional therapeutic strategies to overcome or delay resistance. Here, we identified a pharmacologically targetable metabolic mechanism that drives resistance to EGFR TKIs in lung cancer cell lines and patient-derived xenograft mice. We demonstrated that aldo-keto reductase family 1 member B1 (AKR1B1) interacts with and activates signal transducer and activator of transcription 3 (STAT3) to up-regulate the cystine transporter solute carrier family 7 member 11 (SLC7A11). This leads to enhanced cystine uptake and flux to glutathione de novo synthesis, reactive oxygen species (ROS) scavenging, protection from cell death, and EGFR TKI drug resistance in lung cancer cell lines and xenograft mouse models. Suppression of AKR1B1 with selective inhibitors, including the clinically approved antidiabetic drug epalrestat, restored the sensitivity of resistant cell lines to EGFR TKIs and delayed resistance in lung cancer patient-derived xenograft mice. Our findings suggest a metabolic mechanism for resistance to a molecularly targeted therapy and provide a potential therapeutic target for overcoming resistance to EGFR TKIs, including the third-generation inhibitor osimertinib.

Monitoring PSMA Responses to ADT in Prostate Cancer Patient-Derived Xenograft Mouse Models Using [18F]DCFPyL PET Imaging.

PSMA overexpression has been associated with aggressive prostate cancer (PCa). However, PSMA PET imaging has revealed highly variable changes in PSMA expression in response to ADT treatment ranging from increases to moderate decreases. To better understand these PSMA responses and potential relationship to progressive PCa, the PET imaging agent, [18F]DCFPyL, was used to assess changes in PSMA expression in response to ADT using genomically characterized LuCaP patient-derived xenograft mouse models (LuCaP-PDXs) which were found to be sensitive to ADT (LuCaP73 and LuCaP136;CS) or resistant (LuCaP167;CR). [18F]DCFPyL (2-(3-{1-carboxy-5-[(6-[18F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid) was used to assess PSMA in vitro (saturation assays) in LuCaP tumor membrane homogenates and in vivo (imaging/biodistribution) in LuCaP-PDXs. Control and ADT-treated LuCaPs were imaged before ADT (0 days) and 2-, 7-, 14-, and 21-days post-ADT from which tumor:muscle ratios (T:Ms) were determined and concurrently tumor volumes were measured (caliper). After the 21-day imaging, biodistributions and histologic/genomic (PSMA, AR) analysis were done. [18F]DCFPyL exhibited high affinity for PSMA and distinguished different levels of PSMA in LuCaP tumors. Post-ADT CS LuCaP73 and LuCaP136 tumor volumes significantly decreased at day 7 or 14 respectively vs controls, whereas the CR LuCaP167 tumor volumes were minimally changed. [18F]DCFPyL imaging T:Ms were increased 3-5-fold in treated LuCaP73 tumors vs controls, while treated LuCaP136 T:Ms remained unchanged which was confirmed by day 21 biodistribution results. For treated LuCaP167, T:Ms were decreased (~ 45 %) vs controls but due to low T:M values (<2) may not be indicative of PSMA level changes. LuCaP73 tumor PSMA histologic/genomic results were comparable to imaging/biodistribution results, whereas the results for other tumor types varied. Tumor responses to ADT varied from sensitive to resistant among these LuCaP PDXs, while only the high PSMA expressing LuCaP model exhibited an increase in PSMA levels in response to ADT. These models may be useful in understanding the clinical relevance of PSMA PET responses to ADT and potentially the relationship to disease progression as it may relate to the genomic signature.

Drug resistance-free cytotoxic nanodrugs in composites for cancer therapy.

Drug resistance is a major cause of treatment failure for small-molecule cancer chemotherapies, despite the advances in combination therapies, drug delivery systems, epigenetic drugs, and proteolysis-targeting chimeras. Herein, we report the use of a drug resistance-free cytotoxic nanodrug as an alternative to small-molecule drugs. The present nanodrugs comprise 2 nm core gold nanoparticles (AuNPs) covered completely with multivalent hydrocarbon chains to a final diameter of ∼10 nm as single drug molecules. This hydrophobic drug-platform was delivered in composite form (∼35 nm) with block-copolymer like other small-molecular drugs. Upon uptake by cells, the nanodrugs enhanced the intracellular levels of reactive oxygen species and induced apoptosis, presumably reflecting multivalent interactions between aliphatic chains and intracellular biomolecules. No resistance to our novel nanodrug was observed following multiple treatment passages and the potential for use in cancer therapy was verified in a breast cancer patient-derived xenograft mouse model. These findings provide insight into the use of nano-scaled compounds as agents that evade drug resistance to cancer therapy.

Quantitative structural analysis of glycans expressed within tumors derived from pancreatic cancer patient-derived xenograft mouse models

Pancreatic ductal adenocarcinoma (PDAC) is an intractable malignancy for which novel therapeutic targets are in high demand. To uncover glycans expressed within PDAC, we previously performed glycome profiling of PDAC cell lines using lectin microarray and found that the lectin rBC2LCN with specificity to a Fucα1-2Galβ1-3 motif exhibited strong binding to a PDAC cell line (Capan-1) and to all tumor tissues derived from 69 pancreatic cancer patients. Nevertheless, no information was available as to whether glycans containing the Fucα1-2Galβ1-3 motif are expressed within PDAC. Here we used HPLC combined with MALDI-TOFMS to perform a structural and quantitative glycome analysis targeting both N- and O-glycans derived from two types of patient-derived PDAC xenograft mouse models, PC3 (well-differentiated) and PC42 (poorly-differentiated). A higher percentage of highly branched and sialylated complex-type N-glycans was detected in PC42 relative to PC3. The percentage of core 1 O-glycans was higher in PC42 relative to PC3, whereas that of core 3 O-glycans was higher in PC3. Cancer-related glycan epitopes such as Lewis A and Lewis Y were detected in core 3 O-glycans of both PC3 and PC42. H-type3 containing the Fucα1-2Galβ1-3 motif was detected in Core 2 O-glycans in both models, explaining the molecular mechanism of the binding of rBC2LCN to PDAC.

Antihistamine Drug Ebastine Inhibits Cancer Growth by Targeting Polycomb Group Protein EZH2.

Enhancer of zester homolog 2 (EZH2), a histone lysine methyltransferase and the catalytic component of polycomb repressive complex 2, has been extensively investigated as a chromatin regulator and a transcriptional suppressor by methylating H3 at lysine 27 (H3K27). EZH2 is upregulated or mutated in most cancers, and its expression levels are negatively associated with clinical outcomes. However, the current developed small-molecule inhibitors targeting EZH2 enzymatic activities could not inhibit the growth and progression of solid tumors. Here, we discovered an antihistamine drug, ebastine, as a novel EZH2 inhibitor by targeting EZH2 transcription and subsequently downregulating EZH2 protein level and H3K27 trimethylation in multiple cancer cell lines at concentrations below 10 μmol/L. The inhibition of EZH2 by ebastine further impaired the progression, migration, and invasiveness of these cancer cells. Overexpression of Ezh2 wild-type and its mutant, H689A (lacking methyltransferase activity), rescued the neoplastic properties of these cancer cells after ebastine treatment, suggesting that EZH2 targeted by ebastine is independent of its enzymatic function. Next-generation RNA-sequencing analysis also revealed that C4-2 cells treated with 8 μmol/L ebastine showed a gene profiling pattern similar to EZH2-knockdown C4-2 cells, which was distinctively different from cells treated with GSK126, an EZH2 enzyme inhibitor. In addition, ebastine treatment effectively reduced tumor growth and progression, and enhanced progression-free survival in triple-negative breast cancer and drug-resistant castration-resistant prostate cancer patient-derived xenograft mice. Our data demonstrated that ebastine is a novel, safe, and potent anticancer agent for patients with advanced cancer by targeting the oncoprotein EZH2.

Comparative study on contrast enhancement of Magnevist and Magnevist-loaded nanoparticles in pancreatic cancer PDX model monitored by MRI

BackgroundThe aim of this study was to compare contrast enhancement of Magnevist® (gadopentate dimeglumine (Mag)) to that of PEGylated Magnevist®-loaded liposomal nanoparticles (Mag-Lnps) in pancreatic cancer patient-derived xenograft (PDX) mouse model via magnetic resonance imaging (MRI).MethodsMag-Lnps formulated by thin-film hydration and extrusion was characterized for the particle size and zeta potential. A 21.1 T vertical magnet was used for all MRI. The magnet was equipped with a Bruker Advance console and ParaVision 6.1 acquisitions software. Mag-Lnps phantoms were prepared and imaged with a 10-mm birdcage coil. For in vivo imaging, animals were sedated and injected with a single dose (4 mg/kg) of Mag or Mag-Lnps with Mag equivalent dose. Using a 33-mm inner diameter birdcage coil, T1 maps were acquired, and signal to noise ratio (SNR) measured for 2 h.ResultsMag-Lnps phantoms showed a remarkable augmentation in contrast with Mag increment. However, in in vivo imaging, no significant difference in contrast was observed between Mag and MRI. While Mag-Lnps was observed to have fairly high tumor/muscle (T/M) ratio in the first 30 min, free Mag exhibited higher T/M ratio over the time-period between 30 and 120 min. Overall, there was no statistically significant difference between Mag and Mag-Lnp in rating MR image quality. Low payload of Mag entrapment by Lnps and restricted access of water (protons) to Mag-Lnps may have affected the performance of Mag-Lnps as an effective contrast agent.ConclusionThis study showed no significance difference in MRI contrast between Mag and Mag-Lnp pancreatic cancer PDX mouse models.

Genetic Profiles Associated with Chemoresistance in Patient-Derived Xenograft Models of Ovarian Cancer.

PurposeRecurrence and chemoresistance (CR) are the leading causes of death in patients with high-grade serous carcinoma (HGSC) of the ovary. The aim of this study was to identify genetic changes associated with CR mechanisms using a patient-derived xenograft (PDX) mouse model and genetic sequencing.Materials and MethodsTo generate a CR HGSC PDX tumor, mice bearing subcutaneously implanted HGSC PDX tumors were treated with paclitaxel and carboplatin. We compared gene expression and mutations between chemosensitive (CS) and CR PDX tumors with whole exome and RNA sequencing and selected candidate genes. Correlations between candidate gene expression and clinicopathological variables were explored using the Cancer Genome Atlas (TCGA) database and the Human Protein Atlas (THPA).ResultsThree CR and four CS HGSC PDX tumor models were successfully established. RNA sequencing analysis of the PDX tumors revealed that 146 genes were significantly up-regulated and 54 genes down-regulated in the CR group compared with the CS group. Whole exome sequencing analysis showed 39 mutation sites were identified which only occurred in CR group. Differential expression of SAP25,HLA-DPA1, AKT3, and PIK3R5 genes and mutation of TMEM205 and POLR2A may have important functions in the progression of ovarian cancer chemoresistance. According to TCGA data analysis, patients with high HLA-DPA1 expression were more resistant to initial chemotherapy (p=0.030; odds ratio, 1.845).ConclusionWe successfully established CR ovarian cancer PDX mouse models. PDX-based genetic profiling study could be used to select some candidate genes that could be targeted to overcome chemoresistance of ovarian cancer.

Abstract 9: Efficacy and functional study of tetraarsenic oxide as an anticancer drug in cervical cancer cell lines and cervical cancer patient-derived xenograft mouse

Abstract Background: Arsenic compound has been used as a medicine in China and has been studied to treat acute promyelocytic leukemia, hepatocellular carcinoma, and melanoma. Tetraarsenic oxide (TAO; As4O6) is an inorganic arsenic compound that has been demonstrated to inhibit angiogenesis and cell growth in cervical cancer cells. In this study, we demonstrated anti-cancer effect of TAO in cervical cancer patient-derived xenograft (PDX) mouse model and the functional mechanism related with cervical cancer cell death. Materials &amp; Methods: We used SiHa, Caski, and HeLa cells for cervical cancer cells and HUVEC cells to study functional mechanism of TAO in an in vitro. MTT assay was performed to assess cell death and MMP-2-specific ELISA assay was used to detect MMP-2 expression. To establish the patient-derived xenograft (PDX) mouse model of cervical cancer, surgical patient tumor specimens were reduced to small pieces (less than 2-3 mm), implanted into the subrenal capsules of the left kidneys of BALB/C nude mice, and propagated by serial transplantation. The PDX model used in this study was generated with a tumor that was histologically defined as a FIGO stage Ib1 invasive squamous cell carcinoma. The patient was a 46-year-old woman who received primary debulking surgery followed by radiation therapy for 28 times. Results: We measured IC50 of TAO in cervical cancer and HUVEC cells. IC50 of TAO in SiHa, Caski, and HUVEC was around 3 uM and it was about 0.6 uM in HeLa cells. Although TAO inhibited MMP-2 expression, an important protein for angiogenesis, in both cervical cancer cell lines and HUVEC cells, the functional mechanism was different in between cervical cancer cells and HUVEC cells. TAO inhibited Akt activation in SiHa, Caski, and HeLa. However, TAO had no effect on activation of Akt in HUVEC cells. TAO inhibited expression of VEGF receptor 2 in HUVEC cells but not in cervical cancer cells. We found that TAO inhibited autophagy determined by p62 expression level in cervical cancer cells but not in HUVEC cells. Cervical cancer PDX mouse study demonstrated that TAO inhibited tumor growth. Conclusions: TAO inhibited cell growth of cervical cancer cells and HUVEC. The functional signaling pathway of TAO-induced cell death might different in between cervical cancer cells and endothelial cell, HUVEC. TAO inhibited tumor growth in cervical cancer PDX mouse probably through inhibition of cancer cell growth and inhibition of angiogenesis via inhibition of endothelial cell growth. These results suggest that TAO may be considered as a novel therapeutic compound for the treatment of cervical cancer. Citation Format: Jeong-Won Lee, Jae Ryoung Hwang, Young-Jae Cho, Ji-Hye Kim, Ji Yoon Ryu, E-Sun Baik. Efficacy and functional study of tetraarsenic oxide as an anticancer drug in cervical cancer cell lines and cervical cancer patient-derived xenograft mouse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 9.

PDXliver: a database of liver cancer patient derived xenograft mouse models

BackgroundLiver cancer is the second leading cause of cancer-related deaths and characterized by heterogeneity and drug resistance. Patient-derived xenograft (PDX) models have been widely used in cancer research because they reproduce the characteristics of original tumors. However, the current studies of liver cancer PDX mice are scattered and the number of available PDX models are too small to represent the heterogeneity of liver cancer patients. To improve this situation and to complement available PDX models related resources, here we constructed a comprehensive database, PDXliver, to integrate and analyze liver cancer PDX models.DescriptionCurrently, PDXliver contains 116 PDX models from Chinese liver cancer patients, 51 of them were established by the in-house PDX platform and others were curated from the public literatures. These models are annotated with complete information, including clinical characteristics of patients, genome-wide expression profiles, germline variations, somatic mutations and copy number alterations. Analysis of expression subtypes and mutated genes show that PDXliver represents the diversity of human patients. Another feature of PDXliver is storing drug response data of PDX mice, which makes it possible to explore the association between molecular profiles and drug sensitivity. All data can be accessed via the Browse and Search pages. Additionally, two tools are provided to interactively visualize the omics data of selected PDXs or to compare two groups of PDXs.ConclusionAs far as we known, PDXliver is the first public database of liver cancer PDX models. We hope that this comprehensive resource will accelerate the utility of PDX models and facilitate liver cancer research. The PDXliver database is freely available online at: http://www.picb.ac.cn/PDXliver/

Abstract 4211: Discovery of a novel peptide for improvement of radiotherapeutic efficacy and development of prognotic diagnosis

Abstract Cancer can be treated by the various modalities including surgery, chemotherapy, radiotherapy, among which the radiotherapy is the most important therapeutics in the cancer treatment. Despite the progression of modalities, cancer therapy has been constrained by impasse of prediction of treatment outcome and direction of cancer therapeutics because of diagnosis date, metastasis and genetic diversity of the individual patients. In this study, we discovered specific tumor targeting peptides and radiotherapy-responding peptides suitable to apply to cancer therapy and monitoring of radiotherapy response. For a non-invasive imaging for potential cancer diagnosis system, we screened peptides using the phage display technique. Phage display is used in various pharmaceutical biotechnologies, and the one of the most powerful technologies to identify novel peptides regulating carcinogenesis. To emphasize the function of peptide ligands in a living body and consider the tumoral microenvironment, we used the patient-derived xenograft (PDX) tumor models for in vivo peptide displayed phage screening. It is warranted that the peptides specifically targeting in vivo human cancer xenografts with radiation treatment or not in colorectal cancer PDXs. We performed in vivo peptide screening in the two different cases of colorectal cancer PDX mouse models, and selected peptides were confirmed to radiation responding peptides and radiation resistant peptides. The results suggested that selected peptides were a promising diagnostic lead molecule for rapid and accurate detection for the radiotherapy response. Taken together, we described a strategy to identify the peptides by in vivo phage display, and peptides possessed the specific targeting ability which would be potentially feasible for the development of personalized precision treatment. Its might be potential IR-guided probe and prognostic diagnosis in radiotherapy for colorectal cancer. Keywords: Peptide, Colorectal cancer, Patient-derived xenograft (PDX), Radiation, In vivo peptide screening Citation Format: Kyoung Jin Lee, Jae Hee Lee, Si Yeol Song, Seong-Yun Jeong, Eun Kyung Choi. Discovery of a novel peptide for improvement of radiotherapeutic efficacy and development of prognotic diagnosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4211.