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Published in final edited form as: J Pediatr Surg. 2023 Oct 7;59(3):473–482. doi: 10.1016/j.jpedsurg.2023.10.004

CDK4/6 inhibition with lerociclib is a potential therapeutic strategy for the treatment of pediatric sarcomas

Janet R Julson 1,*, Sara C Horton 2,*, Colin H Quinn 1, Andee M Beierle 3, Laura V Bownes 1, Jerry E Stewart 1, Jamie Aye 2, Karina J Yoon 4, Elizabeth A Beierle 1,†
PMCID: PMC10922146  NIHMSID: NIHMS1936549  PMID: 37919169

Abstract

Background:

Sarcomas are a heterogenous collection of bone and soft tissue tumors. The heterogeneity of these tumors makes it difficult to standardize treatment. CDK 4/6 inhibitors are a family of targeted agents which limit cell cycle progression and have been shown to be upregulated in sarcomas. In the current preclinical study, we evaluated the effects of lerociclib, a CDK4/6 inhibitor, on pediatric sarcomas in vitro and in 3D bioprinted tumors.

Methods:

The effects of lerociclib on viability, proliferation, cell cycle, motility, and stemness were assessed in established sarcoma cell lines, U-2 OS and MG-63, as well as sarcoma patient-derived xenografts (PDXs). 3D printed biotumors of each of the U-2 OS, MG-63, and COA79 cells were utilized to study the effects of lerociclib on tumor growth ex vivo.

Results:

CDK 4/6, as well as the intermediaries retinoblastoma protein (Rb) and phosphorylated Rb were identified as targets in the four sarcoma cell lines. Lerociclib treatment induced cell cycle arrest, decreased proliferation, motility, and stemness of sarcoma cells. Treatment with lerociclib decreased sarcoma cell viability in both traditional 2D culture as well as 3D bioprinted microtumors.

Conclusions:

Inhibition of CDK 4/6 activity with lerociclib was efficacious in traditional 2D sarcoma cell culture as well as in 3D bioprints. Lerociclib holds promise and warrants further investigation as a novel therapeutic strategy for management of these heterogenous groups of tumors.

Keywords: Lerociclib, CDK 4/6 inhibitors, 3D bioprinting, Pediatric sarcomas

1. Introduction

The term sarcoma encompasses a large category of tumors that has been divided into bone or soft tissue sarcomas with numerous further classifications within this division.[1] The mainstay of management of pediatric soft tissue sarcomas includes surgical resection of the primary tumor with neoadjuvant and/or adjuvant chemotherapy and/or radiation.[1] Despite multimodal treatment regimens, prognosis for soft tissue sarcomas remains poor with 5-year survival rates 63–84%[2] which is due, in part, to tumor heterogeneity as well as multiple mechanisms of drug resistance.[1] Efforts to conduct high throughput molecular analysis such as those by the MULTISARC trial highlight the importance of focusing on targeted therapies to improve outcomes for patients with sarcoma.[3]

One potential therapeutic target is the cell cycle regulatory protein, cyclin-dependent kinase 4 (CDK4). CDK4/6 forms a complex with cyclin D1 which phosphorylates the retinoblastoma tumor suppressor protein (Rb). Once phosphorylated, Rb is released from E2F, allowing transcription of cell cycle genes resulting in cell cycle progression from G1 to S phase (Fig. 1A).[4] Several CDK4/6 inhibitors have been developed including the third generation inhibitors, palbociclib, ribociclib, and abemaciclib. These agents have been studied in vitro in other cancer types including esophageal, ovarian, pancreatic, and colorectal cancers as well as glioblastoma, neuroblastoma, and melanoma where they have been shown to decrease tumor cell proliferation, viability, tumor sphere formation, and induce cell cycle arrest both alone and in combination therapy.[5–13] CDK4/6 inhibitors have proven efficacious in clinical trials for breast cancer patients; ultimately leading to Food and Drug Administration for approval of use of palbociclib in that cancer.[14–17] There is evidence that CDK4 inhibition may be useful in sarcoma.[1,15,16,18] Amplification of CDK4 has been noted in sarcoma subtypes[19] as well as alterations in other genes along this pathway including RB1.[20] CDK4 inhibition with palbociclib induced cell cycle arrest in vitro in rhabdomyosarcoma cells[13] as well as in soft tissue and bone sarcoma patient derived xenografts (PDXs).[16] Zhou and colleagues demonstrated that siRNA knockdown of CDK4 decreased osteosarcoma cell proliferation and motility, and induced apoptosis.[18] Additionally, treatment with palbociclib resulted in decreased soft tissue sarcoma patient-derived orthotopic xenograft tumor growth.[21]

Figure 1. Increased expression of CDK4 correlates with worse outcomes for patients with sarcoma.

Figure 1.

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(A) Schematic demonstrating the CDK4/6 cyclin D1 complex which phosphorylates Rb, permitting the release of Rb from E2F. E2F promotes transcription of cell cycle genes and allows progression of the cell cycle from G1 to S phase. (B) Characteristics of the cell lines and PDXs utilized in the current study. (C) Query of the TCGA-SARC database evaluating the level of CDK4 abundance and patient vital status at follow-up. Patients with lower CDK4 abundance (n = 184) were more likely to be alive at follow-up compared to those with higher amounts (n = 75) (p = 0.043). (D, E) Immunoblotting of whole cell lysates confirmed the expression of CDK4 and CDK6 in U-2 OS (D) and COA79 (E) cell lines. Rb protein expression was mostly unchanged after treatment with lerociclib, but phosphorylation of Rb at the S780 and S795 sites was decreased. Vinculin served as an internal loading control.

Shortcomings to the early CDK4/6 inhibitors include the development of inhibitor resistance and significant myelosuppression[14,22,23] leading to the development of new CDK4/6 inhibitors. These newer compounds are cleared from the plasma more quickly, minimizing the effects on the bone marrow and allowing for continuous treatment and reduced risk of drug resistance.[24] G1T38, or lerociclib, is an example of these improved inhibitors. A phase I trial in lung cancer evaluating lerociclib in combination with osimertinib, an EGFR tyrosine kinase inhibitor, found that only 1 of 30 patients enrolled experienced a dose limiting toxicity of neutropenia.[25] Another phase II clinical trial with lerociclib is ongoing, evaluating lerociclib in combination with fulvestrant in breast cancer.[26] Because of the preclinical data suggesting utility of CDK4 inhibition in sarcoma and the interest in lerociclib as an improved agent, in this study, we explore lerociclib as a strategy in the management of sarcomas.

2. Materials & Methods

2.1. Database analysis

R2 (http://r2.amc.nl, accessed on April 8, 2023), a publicly available genomic analysis platform, was utilized to interrogate microarray and RNA-seq data from the cancer genome atlas sarcoma collection (TCGA-SARC)[27] to compare vital status (alive or dead) at follow-up with CDK4 expression. The setpoints which define “high” and “low” expression in the R2 database are based on an algorithm in the database known as Hugo Once. The average expression of an individual gene is determined with each individual gene filter applied (i.e. in this case, patients who have both CDK4 expression measured and also have data to correlate with vital status) after logarithmic transformation and then the appropriate statistical analysis test is conducted within the database so they are not necessarily a set point value - median, minimum group number, etc. (https://hgserver1.amc.nl/r2/help/r2_tutorials.pdf)

2.2. Cell lines and culture

The long-term passage cells lines, U-2 OS and MG-63 were used for these studies. U-2 OS cells were obtained from the American Type Culture Collection (ATCC, HTB-96, Manassas, VA) and represent a moderately differentiated osteosarcoma with epithelial morphology (Fig. 1B). MG-63 cells were obtained from ATCC (CRL-1427) and represent osteosarcoma with fibroblast morphology (Fig. 1B). U-2 OS cells were cultured in McCoy’s 5A medium (Gibco, Grand Island, NY), 10% fetal bovine serum (FBS, Hyclone, Suwanee, GA), 0.22 mg/mL L-glutamine (MP Biomedicals, Solon, OH), 3 mg/mL D-glucose (Sigma Aldrich, Burlington, MA), and 1 μg/mL penicillin/streptomycin (Sigma Aldrich). MG-63 cells were maintained in minimum Eagle medium (Corning Inc., Corning, NY), with 10% FBS (Hyclone), 1 μg/mL penicillin/streptomycin (Sigma Aldrich), 0.3 mg/mL L-glutamine (MP Biomedicals), and 0.11mg/mL sodium pyruvate (Sigma-Aldrich). Cell lines were propagated in sterile incubators at 37 °C with 5% carbon dioxide (CO2).

2.3. Patient-derived xenografts (PDXs)

The PDXs, COA30 and COA79, relapsed synovial sarcoma lung metastasis, were established as previously described[28] and characterized in Figure 1B. Briefly, following written informed consent and under an institutional review board (IRB) approved protocol (University of Alabama at Birmingham (UAB) IRB 130627006), a fresh piece of tumor was excised from a pediatric patient who was undergoing surgical treatment for their tumor. The specimen was placed in iced Roswell Park Memorial Institute 1650 (RPMI) medium. Following an approved UAB institution animal care and use committee protocol, (IACUC 009186), a portion of the tumor was implanted into a female athymic nude mouse (Charles River, Frederick, MD). Briefly, animals were anesthetized with 3% inhalational isoflurane and under sterile conditions, 16 mm3 tumor pieces were injected into the subcutaneous space of the animal’s flank. Animals were housed in a pathogen-free environment where they were monitored for overall health and tumor growth. Tumor volumes were measured weekly and harvested when IACUC parameters were met. For in vitro experiments, tumors were dissociated using a Tumor Dissociation Kit (Miltenyi Biotec, San Diego, CA) per manufacturer’s protocol. They were maintained in neurobasal (NB) medium (Life Technologies) with the addition of B-27 without Vitamin A (Life Technologies), N2 (Life Technologies), L-glutamine (2 mM, Thermo Fisher Scientific Inc.), gentamicin (50 μg/mL, Thermo Fisher Scientific Inc.), amphotericin B (250 μg/mL, Thermo Fisher Scientific Inc.), epidermal growth factor (10 ng/mL, Miltenyi Biotec), and fibroblast growth factor (10 ng/mL, Miltenyi Biotec) and maintained at 37 °C with 5% CO2 overnight prior to utilizing for in vitro experiments previously described.28

Integrity of the cell lines and of the PDXs was insured by yearly short tandem repeat analysis (UAB Genomics Core, Birmingham, AL) and real-time PCR (qPCR) to detect potential murine contamination of the PDX (TRENDD RNA/DNA Isolation and TaqMan QPCR/Genotyping Core Facility, UAB, Birmingham, AL) (human GAPD endogenous control, cat number 4326317E; mouse GAPD endogenous control, cat number 4352339E; ThermoFisher Scientific Inc.). Histology and immunohistochemistry (IHC) confirmed recapitulation of the PDXs with the patient’s tumor. Established cell lines were tested twice yearly and deemed free of Mycoplasma infection by testing with the Universal Mycoplasma Detection Kit (30-1012K, ATCC).

2.4. Antibodies and reagents

Primary antibodies utilized in these studies included the following: CDK4 (12790S), CDK6 (1331S), Rb (9313S), phospho-Rb S780 (9307S), phospho-Rb S795 (9301S), and polyclonal anti-vinculin (4650) obtained from Cell Signaling Technology (Beverly, MA). Lerociclib was purchased from Selleck Chemicals (Houston, TX).

2.5. Immunoblotting

Cells were lysed using radio-immunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors (Sigma Aldrich), phosphatase inhibitors (Sigma Aldrich), and phenyl-methane-sulfonyl-fluoride (Sigma Aldrich). Immunoblotting, gel transfer, and immunodetection were performed as previously described.[29] The Precision Plus Protein Kaleidoscope molecular weight marker (Bio-Rad) confirmed the expected size of target proteins. Antibodies were utilized according to the manufacturers’ recommendations. Vinculin expression was used as an internal control to confirm equal protein loading.

2.6. Cell viability and proliferation

The alamarBlue Cell Viability Assay (Thermo Fisher Scientific) was used to measure viability. U-2 OS (1.5 × 104 cells per well), MG-63 (1.5 × 104 cells per well), COA30 (3 × 104 cells per well) or COA79 (3 × 104 cells per well) were plated in 96-well plates. Established cell lines were allowed to adhere overnight, then treated with lerociclib at increasing concentrations (0–10 μM). PDX cells were plated then subsequently treated with lerociclib at increasing concentrations (0–10 μM). Following 72 hours of treatment, 10 μL of alamarBlue reagent was added to each well and the absorbance was measured at excitation wavelength of 562 nm and emission wavelength of 595 nm using a microplate reader (BioTek Gen5, BioTek, Winooski, VT).

The CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI) was used to measure proliferation. U-2 OS (5 × 103 cells per well), MG-63 (5 × 103 cells per well), COA30 (1 × 104 cells per well), or COA79 (1 × 104 cells per well) were plated in 96-well plates. U-2 OS and MG-63 cells were allowed to attach overnight and treated with lerociclib at increasing concentrations (0–10 μM). COA30 and COA79 cells were plated and then subsequently treated with lerociclib at increasing concentrations (0–10 μM). After 72 hours of treatment, 10 μL of CellTiter 96 reagent was added to each well and the absorbance was measured at 490 nm using a microplate reader (BioTek Gen5).

2.7. Cell cycle

U-2 OS, MG-63, COA30 or COA79 cells (1 × 106) were plated in 6 well plates and treated with lerociclib (0–4 μM) for 24 hours. Cells were lifted, washed with PBS, and fixed in cold 100% ethanol. Cells were then stained with 20 μg/mL propidium iodine (Invitrogen, Thermo Fisher, Eugene, OR) and 0.2 mg/mL RNAse A (Invitrogen) in 0.1% Triton X (Active Motif, Carlsbad, CA). The Attune NxT Flow Cytometer (Invitrogen) was used to obtain data and analysis conducted with FlowJo software (FlowJo, LLC, Ashland, OR).

2.8. 3D bioprinting

Osteosarcoma (U-2 OS and MG-63) and PDX sarcoma (COA30, COA79) cells (1 × 106) were mixed in 100 μL of bioink composed of 1% sodium alginate and 6% gelatin (Provona UV-MVG, Dupont Nutrition Norge As, Sandvika, Norway). The homogenous mixture was loaded into a 3 mL Cellink (Cellink, Boston, MA) printing syringe. A Cellink BIO XTM printer (Cellink) was set to print at a pressure of 10 kPa for a 0.4 second extrusion time through a 22-gauge needle. Individual prints were printed onto a 96-well plate. Calcium chloride (40 μL) was added to each print for five minutes to achieve crosslinking. The crosslinking agent was washed from the prints with 100 μL of sterile PBS and prints placed in 200 μL of their respective media. Prints were incubated at 37 °C in 5% CO2 overnight and then treated with increasing doses of lerociclib (0–25 μM) for 24 to 72 hours. To measure viability, alamarBlue dye (10 μL, Thermo Fisher Scientific) was added. A microplate reader (BioTek Gen 5) measured absorbance at 570 nm, using 600 nm as a reference.

2.9. Motility

Migration and invasion assays were conducted as previously described.[30] Briefly, 8 μm micropore Transwell inserts from 24-well culture plates (Corning Life Sciences) were coated on the bottom with fibronectin (10 μg/mL, Qiagen, Germantown, MD) for U-2 OS, MG-63, and COA79 or human laminin (AG56P, 10 μg/mL, EMD Millipore) for COA30 for 4 hours at 37 °C then washed with PBS. For invasion assays, the top of the well was coated with 50 μL of Matrigel (1 mg/mL, BD Biosciences, San Jose, CA) for 4 hours at 37 °C. U-2 OS (4 × 104), MG-63 (5 × 104), COA30 (1 × 105) or COA79 (1 × 105) cells were plated and treated with increasing concentrations (0–2.5 μM) of lerociclib. Cells were allowed to migrate or invade for 24 hours (U-2 OS), 72 hours (MG-63 and COA79) or 7 days (COA30). The inserts were fixed with 3% paraformaldehyde for 10 minutes and stained with 1% crystal violet for 30 minutes. A light microscope obtained images of the inserts and the number of cells in seven random fields per insert were counted using ImageJ (http://imagej.nih.gov/ij/), accessed June 1, 2021).

To assess motility in MG-63 cells, a monolayer wound healing (scratch) assay was performed. Cells (5 × 104) were plated in 12-well plates and treated for 24 hours with 0–1 μM lerociclib. When cells reached 80% confluence, a sterile 200 μL pipette tip was used to make a standard scratch in the cell layer. Photographs of the plates were obtained at 0, 12, 24, 36 and 48 hours. ImageJ MRI Wound Healing Tool (http://imagej.nih.gov/ij/), accessed June 1, 2021) quantified the open wound area, and data reported as mean fold change of the open area.

2.10. Extreme limiting dilution assay (ELDA)

Non-adherent COA30 and COA79 cells were plated in NB media in 96 well ultra-low attachment plates using serial dilutions from 5000 to 1 cell per well. Treatment media (100 μL) with either 0 or 1 μM lerociclib was added to each well. Using similar non-adherent conditions, U-2 OS and MG-63 cells were plated in serum-free DMEM (Corning Inc.) with B-27 without Vitamin A (Life Technologies), L-glutamine (2 mM, Thermo Fisher Scientific Inc.), epidermal growth factor (10 ng/mL, Miltenyi Biotec), fibroblast growth factor (10 ng/mL, Miltenyi Biotec) and bovine serum albumin (0.4%, Fisher BioReagents, Pittsburgh, PA). For U-2 OS and MG-63 cells, 100 μL of treatment media with either 0 or 2 μM lerociclib was added to each well. After 1 week, a single blinded researcher evaluated the wells for the presence or absence of spheres. Extreme limiting dilution analysis software was used to analyze the data (http://bioinf.wehi.edu.au/software/elda/). Tables demonstrating estimated and 95% confidence intervals for the 1/ (stem cell frequency) for each group were generated (Supp Fig. S1D).

2.11. Data analysis

Experiments were repeated with at least three biologic replicates and data reported as mean ± standard error of the mean (SEM) unless otherwise stated.[31] To determine statistical significance, a Student’s t-test or ANOVA was used, with p ≤ 0.05 considered statistically significant.

3. Results

3.1. Increased expression of CDK4 correlates with worse outcomes for patients with sarcoma.

We utilized data from the publicly available TCGA-SARC database to evaluate whether the level of CDK4 expression was associated with death at time of follow-up. Of the 259 sarcoma patients, those with lower CDK4 expression were more likely to be alive (n = 184) at follow-up compared to those with higher expression (n = 75) (p = 0.043) (Fig. 1C). As CDK4 inhibition has been investigated as a potential therapeutic target in sarcoma,[12,13,18] we next sought to explore the role of CDK4 inhibition in vitro.

3.2. CDK4 is a targetable protein in sarcoma cells.

Immunoblotting confirmed the expression of CDK4, CDK6, and Rb in U-2 OS, COA79 (Fig. 1D, E) and MG-63 (Supp 1A) cell lines. The effect of lerociclib on the function of CDK4/6 was determined by evaluating the phosphorylation of Rb protein. Phosphorylation of Rb by the CDK4/6-cyclin D1 complex allows E2F to function as a transcription factor allowing for transcription of cell cycle genes thereby promoting progression of the cell cycle from G1 to S (Fig. 1A).[32] It has been shown that phosphorylation of Rb at S780 or S795 allows for the dissociation of E2F1 from pRb[33,34] and thus, we chose to focus on these sites. In U-2 OS (Fig. 1D), COA79 (Fig. 1E), and MG-63 (Supp 1A) cells, treatment with increasing concentrations of lerociclib resulted in a decrease in phosphorylation of Rb at the S780 and S795 sites with no change in total Rb protein expression. In the U-2 OS cell line, there was an increase in expression of CDK4 and CDK6 with lerociclib treatment (1–2 μM) (Fig. 1D). MG-63 cells also demonstrated an increase in CDK6 expression with higher levels of lerociclib treatment (4–6 μM) (Supp Fig. 1A).

3.3. Lerociclib treatment decreases proliferation and attenuates cell cycle progression.

With the understanding that lerociclib functions to decrease cell cycle progression by blocking CDK4/6 activity at G1 phase, we next sought to evaluate its effects on proliferation. CDK4/6 inhibition with lerociclib significantly decreased proliferation in U-2 OS cells with a calculated half-inhibitory concentration (IC50) of 4.8 μM (Fig. 2A) with similar results seen in MG-63 cells (IC50 = 5.1 μM, Fig. 2A). In the PDX cells, the IC50 was similar at 4.0 μM in COA30 cells (Fig. 2A) and 3.0 μM in COA79 cells (Fig. 2A).

Figure 2: Lerociclib treatment decreases proliferation and attenuates cell cycle progression.

Figure 2:

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(A) Proliferation was assessed using cell titer assay. U-2 OS (5 × 103 cells per well), MG-63 (5 × 103 cells per well), COA30 (1 × 104 cells per well) or COA79 (1 × 104 cells per well) were plated in 96-well plates, allowed to attach overnight, and treated with lerociclib at increasing concentrations (0–10 μM) for 72 hours. There was a statistically significant decrease in proliferation in all cell lines beginning at 5.0 μM. (B) Flow cytometry evaluated the effects of lerociclib treatment on the cell cycle. U-2 OS, MG-63, COA30 or COA79 cells (1 × 106) were plated in 6 well plates and treated with 0–4 μM lerociclib for 24 hours. Cells were stained with 20 μg/mL propidium iodine and analyzed by flow cytometry. Treatment with lerociclib led to a significant increase in the percentage of cells in G1 phase in U-2 OS, MG-63, and COA79 cells and a decrease in the percentage of cells in S phase in U-2 OS, COA30, and COA79 cells. (C) Cell cycle data presented in graphic form. Bolded numerals indicate statistically significant values. (D) Representative histogram for cell cycle presented. All experiments were repeated with at least three biologic replicates. Data are reported as mean ± SEM. *p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

To further characterize the effects of lerociclib on cell proliferation, we conducted cell cycle analysis. Treatment with lerociclib led to a significant increase in the percentage of cells in G1 phase in U-2 OS (62.0 ± 5.9 v. 31.2 ± 4.8, treated v. untreated, p ≤ 0.01), MG-63 (93.8 ± 1.0 v. 79.2 ± 0.2, treated v. untreated, p ≤ 0.01), and COA79 cells (80.8 ± 0.6 v. 69.6 ± 0.4, treated v. untreated, p ≤ 0.01) (Fig. 2B, C). There was a significant decrease in the percentage of cells in S phase after lerociclib in U-2 OS (22.5 ± 6.4 v. 38.6 ± 3.9, treated v. untreated, p ≤ 0.01), COA30 (11.3 ± 0.5 v. 16.7 ± 1.5, treated v. untreated, p ≤ 0.05), and COA79 cells (9.4 ± 0.7 v. 13.9 ± 1.4, treated v. untreated, p ≤ 0.05) (Fig. 2B, C). The number of cells in S phase in MG-63 was also decreased but did not reach statistical significance (Fig. 2C). Representative histograms of cell cycle are provided in Fig. 2D.

3.4. Lerociclib treatment significantly diminishes sarcoma cell viability.

With the findings of altered cell proliferation and cell cycle progression, we evaluated the effects of lerociclib on sarcoma cell viability. We found that increasing concentrations of lerociclib diminished the viability of all four sarcoma cell lines (Fig. 3A).

Figure 3. Lerociclib treatment affects sarcoma viability in 2D and 3D cultures.

Figure 3.

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Viability was measured using alamarBlue assay. (A) Sarcoma cells were cultured in standard 2D conditions. U-2 OS (1.5 × 104 cells per well), MG-63 (1.5 × 104 cells per well), COA30 (3 × 104 cells per well), or COA79 (3 ×104 cells per well) were plated in 96-well plates, allowed to attach overnight, and treated with lerociclib at increasing concentrations (0–10 μM) for 72 hours. Treatment with increasing concentrations of lerociclib resulted in decreased viability in all four sarcoma cell lines. (B) COA79 cells (1 × 106) were mixed in in 100 μL of 1% sodium alginate and 6% gelatin bioink and printed as a mixed print onto a 96-well plate. Prints were incubated overnight and treated with increasing doses of lerociclib (0–10 μM) for 72 hours. Lerociclib significantly decreased bioprint viability, but the 3D bioprints were less sensitive than the COA79 cells cultured in 2D conditions (A). (C) U-2 OS cells (1 × 106) were mixed in100 μL of 1% sodium alginate and 6% gelatin bioink and printed as a mixed print onto a 96-well plate. Prints were incubated overnight and treated with increasing doses of lerociclib (0–5 μM) for 24 hours. Lerociclib significantly decreased viability. Viability of the U-2 OS 3D bioprints was affected at similar lerociclib concentrations as the U-2 OS cells cultured in 2D conditions. Experiments for A and C were repeated with at least three biologic replicates. Data for B represent two biologic replicates. Data are reported as mean ± SEM. *p ≤ 0.05, ** p ≤ 0.01

3.5. Lerociclib decreases viability of 3D bioprinted sarcoma microtumors.

Traditional 2D culture has several limitations including diminished cell-cell signaling, unrealistic dispersal of drug treatments, and equal distribution of oxygen and nutrients that may make findings difficult to translate to the clinical setting. PDXs may better recapitulate tumor heterogeneity, but engraftment rates for pediatric sarcomas may be less than 50%[35] and the PDX tumors grow irregularly, with some requiring as long as six months to propagate. 3D bioprinting is one method to overcome this dilemma. To assess sarcoma cell viability, we conducted alamarBlue assays on 3D bioprinted microtumors of U-2 OS and MG-63 cells, and the PDX, COA79. We found MG-63 cells were more resistant to lerociclib in the 3D printed model, with a calculated LD50 of 23.9 μM (Supp 1C) compared to 4.7 μM (Fig. 3A) in 2D culture. Similarly, COA79 cells had increased resistance in the 3D print (LD50 = 8.5 μM, Fig. 3B) compared to 2D culture (LD50 = 3.5 μM, Fig. 3A). The 3D printed U-2 OS cells were more sensitive to lerociclib in the 3D print model, with an LD50 of 1.8 μM compared to 6.7 μM in 2D. These data highlight the difficulties encountered when translating findings from 2D culture to the clinical realm.

3.6. CDK4/6 inhibition reduced the metastatic potential of sarcoma cells.

Metastatic disease is the major cause of mortality for many sarcoma patients. The ability of tumor cells to both migrate and invade contributes to disease metastases. It has been shown that CDK4 expression correlates with metastatic disease in patients with osteosarcoma,[18] leading us to evaluate the effects of lerociclib on sarcoma cell motility and invasion. Motility of U-2 OS, COA30, and COA79 cells was assessed using Transwell migration assay. Treatment with 2.5 μM lerociclib led to a statistically significant decrease in U-2 OS cell migration (p ≤ 0.001) (Fig. 4A). Similarly, in the COA30 and COA79 PDX cells, there was a significant decrease in migration with lerociclib treatment (Fig. 4B, 4C). Motility of MG-63 cells was assessed using monolayer wound healing (scratch) assay. Treatment with lerociclib (1 μM) resulted in a significant decrease in fold change in wound healing area (Supp Fig. 1B).

Figure 4: Lerociclib decreases sarcoma cell migration.

Figure 4:

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Migration assays were conducted using 8 μm micropore Transwell inserts from 24-well culture plates coated with fibronectin (U-2 OS, MG-63, and COA79) or human laminin (COA30). U-2 OS (4 × 104), MG-63 (5 × 104), COA30 (1 × 105) or COA79 (1 × 105) cells were plated and treated with lerociclib (0–4 μM) at concentrations below the calculated LD50 and allowed to migrate through the membrane. The inserts were fixed with 3% paraformaldehyde for 10 minutes and stained with 1% crystal violet for 30 minutes. A light microscope was used to obtain images of the inserts and the number of cells in seven random fields per insert were counted using ImageJ. (A) After treatment with 2.5 μM lerociclib, there was a significant decrease in U-2 OS cell migration (1 v. 0.45 ± 0.04, p ≤ 0.0002). (B) In COA30 cells, treatment with increasing concentrations of lerociclib resulted in a significant decrease in migration at both 2 μM (1 v. 0.18 ± 0.05, p ≤ 0.001) and 4 μM (1 v. 0.45 ± 0.04, p ≤ 0.001). (C) Treatment with 1 μM lerociclib resulted in a decrease in motility of COA79 cells (1 v. 0.81 ± 0.05, p ≤ 0.05). All experiments were repeated with at least three biologic replicates. Data are reported as mean ± SEM. Scale bars represent 100 μm. *p ≤ 0.05, *** p ≤ 0.001

For cancer cells to metastasize, they must not only be able to move, but must possess the ability to invade surrounding tissue.[36] In all four cell lines, we observed a statistically significant decrease in invasion following treatment with lerociclib (Fig. 5A–D). These data indicate that lerociclib decreased the metastatic potential of sarcoma cells.

Figure 5: Lerociclib treatment decreases invasion.

Figure 5:

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For invasion assays, the bottom side of the Transwell plate was treated as described for migration and the top of the plate was coated with 50 μL of Matrigel for 4 hours at 37 °C. U-2 OS (4 × 104), MG-63 (5 × 104), COA30 (1 × 105) or COA79 (1 × 105) cells were plated and treated with increasing concentrations (0–2.5 μM) of lerociclib. Cells were allowed to invade through the Matrigel coating, fixed with 3% paraformaldehyde for 10 minutes and stained with 1% crystal violet for 30 minutes. A light microscope obtained images of the inserts and the number of cells in seven random fields per insert were counted using ImageJ. (A) In the U-2 OS cell line, there was a significant decrease in invasion after treatment with both 1 μM (1 v. 0.48 ± 0.04, p ≤ 0.0002) and 2.5 μM (1 v. 0.45 ± 0.12, p ≤ 0.01) concentrations of lerociclib. (B) There was a decrease in MG-63 invasion following 1 μM (1 v. 0.46 ± 0.04, p ≤ 0.001) and 2 μM (1 v. 0.37 ± 0.06, p ≤ 0.001) lerociclib concentrations. (C) In COA30 cells, there was a significant decrease in invasion following treatment with both 1 μM (1 v. 0.37 ± 0.10, p ≤ 0.001) and 2 μM (1 v. 0.38 ± 0.14, p ≤ 0.05) doses of lerociclib. (D) In COA79 cells, treatment with lerociclib significantly decreased invasion at 1 μM (1 v. 0.62 ± 0.14, p ≤ 0.05) and 2.5 μM (1 v. 0.37 ± 0.07, p ≤ 0.01) concentrations. All experiments were repeated with at least three biologic replicates. Data are reported as mean ± SEM. Scale bars represent 100 μm. *p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

3.7. Lerociclib decreases sarcoma cell stemness.

Sarcomas arise from cells of mesenchymal origin and their heterogeneity is hypothesized to be due, at least in part, to tumorigenesis arising at differing stages in the mesenchymal stem cell development.[37] These tumor-initiating cells, or cancer stem cells, play a number of roles in tumorigenesis such as evading apoptosis, altering cellular metabolism, and promoting drug resistance.[37] Patients with sarcomas who have higher expression of stemness genes have an increased likelihood of metastatic disease and worse outcomes.[38] To evaluate whether CDK4/6 inhibition could reduce stemness in sarcomas, we evaluated tumorsphere formation after treatment with lerociclib. Using an ELDA, we found that treatment with lerociclib led to decreased sphere formation in each of the four sarcoma cell lines compared to untreated controls (Fig. 6A–D) suggesting a decrease in cancer cell stemness.

Figure 6: Treatment with lerociclib decreases sarcoma stemness.

Figure 6:

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Extreme limiting dilution assays were conducted under non-adherent conditions using serial dilutions from 5000 to 1 cell per well to assess the ability of sarcoma cell lines to form spheres after treatment with lerociclib. (A) U-2 OS and (B) MG-63 cells were plated in serum-free DMEM with B-27 without vitamin A, L-glutamine, epidermal growth, fibroblast growth factor, and bovine serum albumin. Treatment media (100 μL) with either 0 or 2 μM lerociclib was added to each well. After 1 week, a single blinded researcher evaluated the wells for the presence or absence of spheres and extreme limiting dilution analysis software was used to analyze the results. There was a statistically significant decrease in the ability of U-2 OS (p ≤ 0.001) (A) or MG-63 (p ≤ 0.001) (B) cells to form spheres. Non-adherent (C) COA30 and (D) COA79 cells were plated in NB media in 96 well ultra-low attachment plates using serial dilutions from 5000 to 1 cell per well. Treatment media (100 μL) with either 0, 1 or 2 μM lerociclib was added to each well. After 1 week, a single blinded researcher evaluated the wells for the presence or absence of spheres and extreme limiting dilution analysis software was used to analyze the results. Lerociclib treatment reduced sphere forming ability in COA30 (p ≤ 0.001) (C) and COA79 (p ≤ 0.001) (D) cells. All experiments were repeated with at least three biologic replicates. Solid lines represent sphere forming capacity with no lerociclib treatment. Dashed lines represent sphere forming ability after lerociclib treatment.

4. Discussion

The treatment of sarcomas remains a therapeutic challenge for a multitude of reasons including low prevalence and an immense disease heterogeneity.[39] The identification of new targets is hindered by a lack of adequate in vivo and in vitro models. A review by Kondo et al. determined that of the roughly 120 sarcoma subtypes characterized by the WHO, only 45 subtypes had corresponding cell lines, with Ewing sarcoma (156 cell lines) and osteosarcoma (148 cell lines) being the most common.[40] In the current study, we utilized synovial sarcoma PDXs, and it is worth noting that there are only 5 publicly available synovial sarcoma cell lines, highlighting the novelty of our findings.[40]

PDXs are frequently used to complement the findings seen using established cell lines. The first PDX model was established over 50 years ago by Rygaar and Povlsen.[41] A review of the Patient Derived Cancer Models repository revealed 168 recorded sarcoma PDX models, though only 5 are synovial sarcoma.[42] Conducting research with these models may be hampered by low rates of engraftment[43,44] and in our experience, variable growth rates necessitating up to six months for tumor growth. A six month lag time is insufficient for treating a lethal disease and makes the investigation of new compounds difficult, leading us to choose 3D printing for the current study.

There are changes in the tumor microenvironment, including signaling between cells and alterations in cellular nutrient and oxygen distribution, that are better captured in 3D culture.[45] Towards this aim, Bruland et al. described the ability to generate sarcoma spheroids from sarcoma cell lines.[46] Patient-derived sarcoma organoids have been shown to grow when injected into an animal model, maintain histologic structure and characteristics of their tumor of origin, and serve as models for chemotherapeutic testing.[47] Limitations of organoid models include determining the ideal culture environment and issues with the Matrigel required for organoid growth affecting tumor cell differentiation and proliferation.[45] These limitations prompted our exploration of 3D bioprinting.

Over the past fifteen years, a number of methods and materials for bioprinting in cancer models have been described.[48] Several tumor types have been studied including skin, liver, ovary, lung, pancreas, cervix, colorectal, and breast cancers, as well as brain and bone tumors.[49] While 3D bioprinted models of bone tumors exist, most address metastatic disease from breast cancer rather than primary bone tumors.[50–53] There have been descriptive reports of bioprinting the osteosarcoma cell lines SaOS-2[51,54,55] U-2 OS,[56] MG-63,[57] as well as the Ewing sarcoma line, TC71,[58] and one patient derived sarcoma organoid.[59] To our knowledge, the current study is the first to describe 3D bioprints of human sarcoma PDX lines.

In the current study, the 3D printed models of MG-63 and COA79 cells were more resistant to lerociclib than those in 2D culture, but the U-2 OS cells were more sensitive compared to 2D cultured cells. Other authors have had similar findings. Pelligrini et al. explored a 3D bioprint model for drug testing using U-2 OS cell lines and found higher expression of drug-resistance markers in cells cultured in 3D compared to those in 2D conditions, even when comparing cisplatin resistant and cisplatin sensitive U-2 OS cell lines.[56] Cisplatin and lerociclib have vastly different mechanisms of action, perhaps explaining the differences in the sensitivities in 2D versus 3D conditions between our studies, but the findings in the current study highlight the difficulties involved in directly translating findings in 2D to the clinical situation. These findings also emphasize the difficulty encountered in prescribing a single agent to combat solid tumors and highlight the importance of considering innovative agents as adjuncts to current therapies.

In the current study, we used changes in phosphorylation of Rb protein as a marker of lerociclib target engagement. Following lerociclib treatment, we did observe decreased phosphorylation of Rb protein, but it was accompanied by increased expression of CDK4/6 in some of the cell lines. Other investigators have noted similar findings. Studies of abemaciclib in breast cancer showed an increase in both CDK6 gene and CDK6 protein expression after treatment,[59] but these changes were not associated with a change in phospho-Rb, suggesting chemotherapeutic resistance to this pathway, which was not seen in our studies with lerociclib. Other investigators have demonstrated CDK4/6 inhibition through upregulation of D-type cyclins, TP53, and the PI3K/AKT/mTOR pathway.[60] Further investigating the potential mechanisms of lerociclib resistance in these pathways will be the direction of future investigations as drug resistance is of great importance in finding better suited therapies for treating sarcomas.

Exploring the role of lerociclib in sarcoma cell motility was a novel aspect of this study. Zhou et al. studied the effects of CDK4/6 inhibition via palbociclib in vitro and found significantly delayed wound healing time in U-2 OS and KHOS osteosarcoma cells.[18] Similarly, palbociclib treatment in the synovial sarcoma cell lines, SYO-1 and Fuji, resulted in a significant decrease in cell motility as measured by wound healing assay.[61] Using migration and invasion assays, we found concordant findings not just in long term passage cells as others have described, but also in the PDXs. The effects on motility are important, as metastatic disease is the predominant cause of mortality in patients with sarcomas.

Conclusion

In this study, we demonstrate that CDK4/6 inhibition with lerociclib is a potential therapeutic adjunct for the treatment of pediatric sarcomas. We also describe the novel technique of 3D bioprinting sarcoma PDX cell lines. We envision that this technology will allow for more rapid target screening and drug selection for the treatment of a rare and heterogenous disease which is currently lacking in suitable therapeutic options.

Supplementary Material

1. Supplemental Figure 1: Lerociclib targets Rb protein phosphorylation.

(S1A) Immunoblotting confirmed the expression of CDK4/CDK6, a target of lerociclib, in MG-63 cells. Treatment with increasing doses of lerociclib resulted in a decrease in the active form, pRb (S780), without a change in total Rb protein. Vinculin served as an internal loading control. (S1B) Wound healing (scratch) assay was utilized to evaluate the effects of lerociclib on MG-63 motility. Cells (5 × 104) were plated and treated for 24 hours with 0 or1 μM lerociclib. Photographs of the plates were obtained over the course of 48 hours and the open wound area was quantified. There was a significant decrease in MG-63 cell motility over time (1 v. 0.30 ± 0.15, at 48 hours, p ≤ 0.01). (S1C) MG-63 cells (1 × 106) were mixed in in 100 μL of 1% sodium alginate and 6% gelatin bioink and printed as a mixed print onto a 96-well plate. Prints were incubated overnight and treated with increasing doses of lerociclib (0–25 μM) for 72 hours. 3D printed MG-63 cells were not sensitive to lerociclib in concentrations up to 25 μM. (S1D) Table demonstrating estimated and 95% confidence intervals for the 1/ (stem cell frequency) for sphere formation assay.

Highlights:

  • Sarcomas are a family of tumors which remain difficult to treat due to their heterogeneity and lack of adequate cell lines and models for study, especially in the pediatric population.

  • CDK 4/6 inhibition with agents such as lerociclib represent an example of targeted therapies amenable to high throughput testing via novel techniques such as 3D bioprinting.

Acknowledgements

The authors wish to thank Vidya Sagar Hanumanthu from the UAB Comprehensive Flow Cytometry Core.

Funding

This project was made possible by funding from the National Cancer Institute of the National Institutes of Health under award numbers T32 CA229102 (JRJ and LVB), 5T32GM008361 (CHQ), U01 336623.01.05.2026199.10 (AMB), and P30 AR048311 and P30 AI2766 (UAB flow cytometry core). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Other funding sources include Dixon Pediatric Research Foundation (SCH), Hyundai Hope on Wheels, Rally Foundation for Childhood Cancer Research (EAB), Sid Strong Foundation, Elaine Roberts Foundation, Open Hands Overflowing Hearts, Sunshine Consortium for Pediatric Sarcoma Research (EAB, JA).

Footnotes

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Associated Data

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Supplementary Materials

1. Supplemental Figure 1: Lerociclib targets Rb protein phosphorylation.

(S1A) Immunoblotting confirmed the expression of CDK4/CDK6, a target of lerociclib, in MG-63 cells. Treatment with increasing doses of lerociclib resulted in a decrease in the active form, pRb (S780), without a change in total Rb protein. Vinculin served as an internal loading control. (S1B) Wound healing (scratch) assay was utilized to evaluate the effects of lerociclib on MG-63 motility. Cells (5 × 104) were plated and treated for 24 hours with 0 or1 μM lerociclib. Photographs of the plates were obtained over the course of 48 hours and the open wound area was quantified. There was a significant decrease in MG-63 cell motility over time (1 v. 0.30 ± 0.15, at 48 hours, p ≤ 0.01). (S1C) MG-63 cells (1 × 106) were mixed in in 100 μL of 1% sodium alginate and 6% gelatin bioink and printed as a mixed print onto a 96-well plate. Prints were incubated overnight and treated with increasing doses of lerociclib (0–25 μM) for 72 hours. 3D printed MG-63 cells were not sensitive to lerociclib in concentrations up to 25 μM. (S1D) Table demonstrating estimated and 95% confidence intervals for the 1/ (stem cell frequency) for sphere formation assay.

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