Combination Therapy of CDK4/CDK6 Inhibitor Palbociclib and Irradiation in non-Malign and Malign Cells

Background: The CDK 4/6 kinase inhibitor palbociclib is approved for rst line treatment of metastatic breast cancer in combination with hormonal therapy and investigated within clinical trials for melanoma. In metastatic disease frequently palliative radiation treatment is necessary. Due to recent ndings of radiosensitizing effects of palbociclib in HNSCC and HCC, a possible inuence of palbociclib on radiosensitivity was studied in malign and non-malign cells. Methods: Different tumor cells and primary broblasts were treated with palbociclib and ionizing radiation (IR) in 2D and 3D cell cultures. Clonogenic assays were performed to study the sensitizing effect of palbociclib on irradiation induced cytotoxicity. Apoptosis, necrosis and cell cycle distribution was analyzed by ow cytometry. Cell migration was studied with scratch assays. Results: The effect of irradiation on skin cancer cells could be slightly decreased by palbociclib in four out of eight cells comparing 2 Gy vs. 2 Gy + 1 µmol/L in the 2D cell culture model. The 3D cell culture model detected differences in cell death more sensitive than the 2D model. Necrosis was increased in primary broblasts (0.2 µmol/L + IR vs. IR; p = 0.0041), whereas apoptosis was decreased in skin cancer cells ICNI (0.2 µmol/L + IR vs. IR with p = 0.0005 // 2 µmol/L + IR vs. IR with p = 0.0072). Overall palbociclib attenuated radiation induced cell death in skin and breast cancer cells. In malign cells combination therapy seems to rise senescence in clonogenic assays, whereas healthy controls showed more cell death. Palbociclib did not inuence cell cycle distribution in the broblasts but led to an accumulation of the tumor cells in G0/G1 cell cycle phase. Additionally, palbociclib decreased migration behavior of the malign and non-malign cells, but without inuence on e-cadherin expression during inhibitor treatment. Conclusions: Palbociclib increases the radiosensitivity of primary broblasts. In cancer cells palbociclib seems to be radioprotective. This effect in cancer cells is probably based on the accumulation of cells in the less radiosensitive G0/G1 cell cycle phase. Further studies are needed to assess whether a combined therapy of palbociclib and radiation is appropriate.


Introduction / Background
Targeted therapy has replaced standard chemotherapy in several of different tumor entities. This latest available therapy uses speci c cancer characteristics (e.g. mutations) to block or inhibit them and deprive the tumor of its survival advantage. Genes or proteins, ensuring the malign cells to proliferate and survive, can harbor these mutations and therefore hold as a permanent proliferation signal, a mechanism to block apoptosis or supply other survival advantages declared as "hallmarks of cancer" by Hanahan and Weinberg 2000. Nowadays, there are 37 FDA-approved kinase inhibitors (KI) available and over 250 inhibitors are currently studied in clinical trials (Wu et al. 2015). Even though cancer patients respond quite well to kinase inhibitors initially, disease progression and formation of metastases occur too often.
Kinase inhibitors can in uence effectiveness of irradiation by increasing or decreasing cellular responses.
Central nervous system (CNS), spinal and other symptomatic metastases require radiation therapy.
However, kinase inhibitors like e.g. the V600E-mutation-speci c BRaf-inhibitor vemurafenib can act as radiosensitizer and cause serious side effects when using the kinase inhibitor simultaneously with irradiation, whereas dabrafenib causes radiosensitization to a much smaller degree (Hecht et al. 2015; Hecht, Meier et al. 2018). Recent data for combination of stereotactic body irradiation and BRaf-inhibitor treatment shows that it is advisable to pause inhibitor treatment during radiotherapy (Kroeze et al. 2019; Hermann, Christiansen 2019). Therefore, the need arises to study interactions between kinase inhibitors and irradiation to answer the question if a simultaneous treatment is advisable.
From 2015 to 2017 around 607,960 new breast cancer cases were diagnosed (American Cancer Society, Inc., Surveillance Research) and 13 % of them were treated with palbociclib (Kish et al. 2018). Approximately 30 % of all early-state breast cancer patients developed advanced or metastatic disease, which often leads to a radiation therapy (O'Shaughnessy 2005). Since 2015, palbociclib is approved by the FDA as a treatment option for ER + , HER2advanced or metastatic breast cancer. It binds potently and highly selectively to the cell cycle regulation proteins Cyclin-dependent kinase 4 (CDK4) and Cyclindependent kinase 6 (CDK6). Therefore, the phosphorylation of the Retinoblastoma protein Rb is blocked and the cell cycle arrested. The tumor cells are forced in a G1-arrest (Saab et al. 2006). Cells rely on proteins like CDK4 to drive the cell cycle. It was tested in various clinical trials for advanced and metastatic melanoma. Since the activity of CDK4 is increased in melanomas mainly due to loss of functional p16 INK4A as a result of gene deletion, promoter methylation or genetic mutation (The Cancer Genome Atlas Network. Cell 2015), we have focused on working on melanoma cells in addition to breast cancer cells (Martin et al. 2018). Former studies showed that palbociclib increases radiosensitivity in HPV-negative head and neck squamous cell carcinoma (HNSCC) (Göttgens et al. 2019) and hepatocellular carcinoma (HCC) (Huang et al. 2018). Several ndings suggest that palbociclib delay the DNA repair via ATM and p53 pathways, which impede handling with radiation (Hashizume et al. 2016; Fernández-Aroca et al. 2019). The clinical relevance of addressing the question if palbociclib in uences the effect of irradiation at cellular level rises when patients under KI therapy progress or metastases and the need for an additional radiation therapy occurs. Thus, it is necessary to study whether the kinase inhibitor modi es the irradiation effect. We used different tumor and normal tissue cells and commercial cell lines to study a possible in uence of a combination of palbociclib therapy with irradiation. and MCF-7 (ER + , PR + ) were used. MDA-MB-231 and MCF-7 were purchased by CLS cell lines service (Eppelheim, Germany). Primary human melanoma cells (from primary tumors) were collected in the Department of Dermatology of the University Hospital of Erlangen following approval by the institutional review board. Single cell suspensions were generated by digesting tissue samples with collagenase (Sigma Aldrich, München, Germany), hyaluronidase (Sigma Aldrich, München, Germany), and DNAse (Roche, Mannheim, Germany) (Walter & Heinzerling 2018) (Ethic approval no. 204 17 BC). The primary human broblasts SBLF7 and SBLF9 were isolated via skin biopsy of the cutis and subcutis after local anesthesia as described previously (Hecht, Harrer et al. 2018). Brie y, each biopsy was dissected in small pieces, placed in tissue culture asks and each covered with a drop of F-12 medium (Gibco, Waltham, USA) supplemented with 40 % fetal bovine serum (FBS) (Merck, Darmstadt, Germany). After the skin pieces had attached to the culture asks and the rst broblasts had grown out, they were covered with F-12 medium supplemented with 12 % FBS, 2 % non-essential amino acids (NEA) (Merck, Darmstadt, Germany) and 1 % penicillin/streptomycin (Gibco, Waltham, USA). When the primary cells were approximately 80 % con uent they were detached with 0.5 % Trypsin (Gibco, Waltham, USA) and further cultured in the medium mentioned above. For continuous cell culture, broblasts were cultured in F-12

Cell cycle
Seeding and treatment procedure was identical to apoptosis and necrosis analysis. After harvesting, cells were xed in 10 mL of 70 % ethanol (Roth, Karlsruhe, Germany) and 1 mL of serum-reduced cell culture medium for a minimum of 12 hours at + 4 °C and then stained with Hoechst 333258 (Invitrogen, Eugene, USA) for 60 minutes on ice. Cells were analyzed in the Cyto ex ow cytometer. In general, cells need around 24 hours to go through cell cycle. To clearly identify any changes in cell cycle distribution, like G0/G1 or G2/M arrest, treatment of 48 h could be advisable. To test whether 24 h or 48 h of treatment should be done, we tested three cell lines previously (data shown in supplements Figure S1) and performed all experiments later on with 48 h of treatment.

Scratch assay
The migration assay was used to evaluate the migratory ability of breast cancer cell lines MDA-MB-231 and MCF-7. As a control healthy donor skin broblasts (SBLF9) were used. Cells were seeded and incubated to reach a con uence of about 90 % at 37 °C. To minimize the effect of scratch closing through proliferation we starved our cells for 24 hours with 2 %-FBS-medium and exchanged the medium in the beginning of each experiment with fresh 2 %-FBS-medium again. The monolayers in each cavity were then scratched with 10 μL-pipet tip. Cells were treated with none or 10 μmol/L of the inhibitor palbociclib and were irradiated with either 0 Gy or 2 Gy. To record the migration, images of the same area of the cell layer were repeatedly acquired with a microscope (Zeiss Primo Vert, Oberkochen, Germany) at 100x magni cation over different periods of time, but always at 24 h and 48 h. The remaining area of the cell scratch was computed by an image analyzing software (Biomas, MSAB, Erlangen, Germany). A smoothing algorithm ltered the image and set the area at point 0 to 100 % and calculated the remaining areas (Wichmann et al. 2015).

Colony forming assay
Regularly splitted cells were seeded in 6-well-plates with a density of 100 -2000 cells per well. Cells were treated with different concentrations of inhibitor and irradiated after 3 h with 0, 2 or 4 Gy. After another incubation phase of 24 h medium was exchanged by fresh standard medium without any drug and the inhibitor was washed out. Plates were incubated for 10 to 14 days until colonies of minimum 50 cells were developed. Colonies were stained with methylene blue (#66725, Sigma Aldrich, München, Germany) for 30 minutes at room temperature and counted when dry.

Statistics
Statistical analysis was performed using GraphPad Prism 8 software. All experiments were carried out with n = 3 or n = 4 (as declared in associated gure description). Data is presented as mean ± SD. Differences in mean values between multiple groups were analyzed using one/two-tailed Mann-Whitney-U test. P-value ≤ 0.05 was determined as signi cant.

Apoptosis and Necrosis (Annexin V, 7-AAD)
Kinase inhibitors can in uence effectiveness of irradiation by increasing or decreasing cellular responses. We studied whether palbociclib is able to change cellular mechanisms like cell death or cell cycle. Therefore, apoptosis and necrosis induction by combined treatment of the kinase inhibitor palbociclib (3dimensional structure shown in Figure 1A) and IR was studied 48 h after treatment. A previous doseescalation study helped us to gure out the relevant KI concentration. Therefore, cells were treated with 2 nmol/L up to 10  . Skin broblasts (SBLF7, SBLF9) of healthy donors were used as a control. In all studied tumor cells (2-dimentional) there was no difference between the inhibitor treatment with or without 2 Gy IR. Combined therapy tends to increase apoptotic and necrotic rates in both controls and in malign HV18MK, whereas LIWE and ICNI showed no in uence of additional palbociclib treatment (2 Gy + 1 µM) ( Figure 1C). In SBLF7, ANST and HV18MK, the highest inhibitor concentration (10 µmol/L) leads to an apoptotic population of more than 20 % in contrast to all other cell cultures. Treatment of both breast cancer cell lines lead to dramatic increase of necrosis (55 -89 %). In general, healthy broblasts tended to show a decrease of cell death comparing 2 Gy to 2 Gy + 1 µmol/L, in contrast to malign cells which tended to show no in uence or increase of combination therapy. Since the malign skin cancer cells were all isolated from different patients with diverse mutation pro les, it is plausible that these cells behave differently.

3D cell culture
A 3-dimensional cell culture model was established and used to explore the impact on apoptosis and necrosis of palbociclib and IR. We used healthy donor skin broblasts as control, both breast cancer cell lines and one BRaf-wt and one BRaf-V600E-mutated cells (ICNI, Figure 2A). After four days healthy broblasts SBLF7, the melanoma cells ICNI ( Figure 2B) and breast cancer cell lines MDA-MB-231, MCF-7 and formed stable spheroids with diameters between 200 µm and 600 µm ( Figure 2B). BRaf-wt cells ANST did not form spheroids within 96 hours.
ICNI, MDA-MB-231, MCF-7 and SBLF7 were able to form stable spheroids within four days, even under combined treatment of kinase inhibitor and IR ( Figure 2B). Necrotic cells increased in healthy skin broblasts after 0.2 µmol/L inhibitor and combined 2 Gy IR treatment compared to IR alone (p = 0.0041). In contrast, the amount of apoptotic cells was clearly decreased in malign cell culture ICNI by 2 Gy irradiation and combined with 0.2 µmol/L or 2 µmol/L palbociclib (p = 0.0005 and p = 0.0072) ( Figure  2C).

Colony forming assay (cell survival)
Since radiation can not only trigger apoptosis or necrosis, it also can lead to a senescence in malign and non-malign cells. Therefore, we used a colony forming assay as the gold standard for radiosensitivity to measure cell survival during different treatment conditions. Cells were seeded in 6-well-plates, treated for 24 h and incubated for 10 up to 14 days until colonies emerged. We measured cell survival fraction (sf) for different inhibitor concentrations (0.5 µmol/L, 1 µmol/L palbociclib) and different irradiation schemes (0, 2 or 4 Gy).

Cell cycle (Hoechst)
The kinases CDK4 and CDK6 are responsible to overcome the G1-checkpoint and to reach the S-phase of cell cycle ( Figure 4A). Since palbociclib is a CDK4/CDK6 inhibitor we performed a cell cycle analysis to nd differences in the distribution of cell cycle phases depending on inhibitor treatment. Furthermore, it is of clinical interest if kinase inhibitors lead to an increase of cells in G2/M phase, which is known to be more radiosensitive and therefore more bene cial during irradiation as a cancer therapy. In general, cells need around 24 hours to go through cell cycle. To clearly identify any changes in cell cycle distribution, like G0/G1 or G2/M arrest, treatment of 48 h could be advisable. To test whether 24 h or 48 h of treatment should be done, we tested three cell lines previously (data shown in supplemental material Figure S1) and performed all experiments later on with 48 h of treatment. Cells were harvested after treatment, and cell DNA was stained by Hoechst 333258 and analyzed by ow cytometry. Representative DNA histograms of the breast cancer cell line MCF-7 under palbociclib and different irradiation schemes are depict in Figure  4B.
To get an adequate overview of cellular response to palbociclib we tested a range from 2 nmol/L -10 µmol/L for cell death and from 1 µmol/L up to 10

Migration (Scratch assay)
Despite effects on apoptosis and necrosis of palbociclib in malign cell cultures it is interesting to see if there is an in uence on migration or invasion. Scratch assays were performed to study the in uence of palbociclib on breast cancer cell lines MDA-MB-231 and MCF-7 and healthy donor skin broblasts ( Figure  5A -5D). Our previous ndings based on cell death FACS analysis led us to decide to run further experiments with a concentration of 10 µmol/L palbociclib. For cell death analysis we were able to found moderate effects without extreme death rates.
The scratch area using the breast cancer cell line MCF-7 was not closed within 48 h in the untreated control. Irradiation of 2 Gy did in uence the migration (5 % vs. 39 % at 24 h, p = 0.0286). MDA-MB-231 migrated fastest and the scratch area was closed without treatment or 2 Gy IR alone within 22 h. Combined treatment leads to an inhibition of migration and scratch area was closed after 48 h ( Figure   5E), whereas palbociclib monotherapy leads to loss of scratch closing within 48 h signi cantly (0 % vs. 22 %, p = 0.0500). MCF-7 treated with 10 µmol/L palbociclib distinctly inhibited migration (10 % vs. 72 % at 48 h, p = 0.0286) and the closing of the scratch within 48 h was lost ( Figure 5F). Comparing 10 µmol/L to 10 µmol/L + 2 Gy IR showed no difference (72 % vs. 72 %, p = 0.8857).Healthy donor skin broblasts migrated slowest and scratch area was not closed within 48 h even without inhibitor or irradiation treatment. Addition of 10 µmol/L inhibitor leads to a reduction of migration (0 Gy: 76.3 % vs. 106.1 %, p < 0.001; 2 Gy: 67.6 % vs. 85.7 %, p = 0.11), hardly in uenced by irradiation ( Figure 5G). To check if the differing migration behavior is driven by varying expression of E-cadherin we used immuno uorescence for further examination.

Expression of E-cadherin
Different migration behavior in the scratch assays prompted us to study the expression of E-cadherin, a calcium-dependent cell adhesion protein. Breast cancer cell lines MCF-7 and MDA-MB-231 and healthy control cells SBLF9 were used for E-cadherin immunostaining ( Figure 6).
Comparing the untreated breast cancer cell lines, MCF-7 ( Figure 6A) express E-cadherin more intense than MDA-MB-231 ( Figure 6B). E-cadherin is essential for strong adhesion to other cells and matrix components, therefore a higher expression could be an explanation for slower migration like MCF-7 showed in the scratch assay. Untreated primary broblasts SBLF9 ( Figure 6C) express lowest E-cadherin.
Treating cells with either palbociclib, irradiation or a combination of both for 48 h showed no variations in E-cadherin expression (data shown in supplemental gure S4).

Discussion
As mentioned before palbociclib is proven to increase radiosensitivity in e.g. HNSCC and HPC (Göttgens First, we tested a range of increasing palbociclib concentrations from 2 nmol/L up to 10 µmol/L in our cell death FACS protocol. This allowed us to cover a range from a minor response (using 1-2 µmol/L) to a strong response (10 µmol/L) of cells treated with KI w/o IR. Based on our dose escalation study we run further experiments with 1 µmol/L -10 µmol/L because no effects were detected with lower concentrations. For more sensitive clonogenic assays we used 0.5 µmol/L -1 µmol/L, which is close to the serum plasma concentration of 0.4 µmol/L (Tamura et al. 2016). The effect of irradiation on melanoma cells could be slightly decreased by the CDK 4/6 kinase inhibitor palbociclib in four out of eight cells comparing 2 Gy vs. 2 Gy + 1 µmol/L. Decreasing apoptosis and necrosis rates indicated a radioprotective effect by palbociclib in cancer cells, whereas healthy broblasts seem to be more sensitive to combined treatment of both inhibitor and IR than IR alone. That suggests a smaller effect on the tumor than on non-neoplastic tissue in patients, which could lead to increased side effects. Finally, FACS analysis of apoptosis and necrosis in ten different cell lines representing three different subgroups (healthy tissue, breast cancer, skin cancer) seem to be distinctly. The leading cause for these differences could be due to the fact that the six tested skin cancer cells representing cells from different patient biopsies. Different mutation pro les of these patients could lead to such diverse behavior.
Furthermore, we established a 3D hanging drop system in our lab and generated cancer spheroids of different cell lines to test them also in FACS analysis for cell death. A 3-dimensional cell culture system is considered to be less arti cial because of a more tissue-like architecture and therefore zones of varying proliferation and nutrition supply (Edmondson et al. 2014). Finally, our 3D cell culture model was able to con rm the results of the 2D examination and detected differences in cell death more sensitive than the 2D model. Necrotic cells increased in healthy skin broblasts (0.2 µmol/L + 2 Gy IR treatment vs. IR; p = 0.0041). In contrast, the amount of apoptotic cells was clearly decreased in malign cell culture ICNI comparing 2 Gy to 0.2 µmol/L + 2 Gy and 2 µmol/L + 2 Gy (p = 0.0005 and p = 0.0072).
Besides analyzing apoptosis and necrosis inducing effects of palbociclib and irradiation, it is important to investigate another major consequence of irradiation, senescence. Since the gold standard for cell survival testing is the colony formation test, we have examined cells accordingly. After testing a wider range of palbociclib concentrations, we focused here on a concentration of 0.5 µmol/L and 1 µmol/L to treat cells for 10 to 14 days that are more compatible, in contrast to 48 h-treatment in our FACS analysis ( 1 -10 µmol/L). All cell lines showed a signi cant reduction of the survival fraction after 0.5 µmol/L or 1 µmol/L palbociclib treatment (p = 0.05) comparing AUC (area under curve). Nevertheless healthy broblasts SBLF7, as our control for normal tissue, reacted the strongest to irradiation, kinase inhibitor treatment and the combination of both. These results support the conclusion of our cell death (FACS) analysis that the healthy broblasts seem to more sensitive to combination therapy than the malignant cells. However, it also showed up that treating SBLF7 with 0.5 µmol/L + 4 Gy and SBLF9 with 1 µmol/L + 4 Gy seemed to rise the survival fraction in a radioprotecting pattern. Whereas colony forming of ANST (0.5 µmol/L + 4 Gy), RERO (1 µmol/L + 4 Gy) and especially ARPA (1 µmol/L + 4 Gy) decreased evidently indicating a radiosensitizing effect. Our cell death and clonogenic experiments suggests that healthy tissue broblasts are triggered more to die via apoptosis and necrosis after treating them with kinase inhibitor and irradiation. In contrast, malign cells showed reduced survival fractions after combination therapy in our colony forming assays, which could indicate an increase of senescence. Because cancer cells often deregulate their cell death pathways to proliferate uncontrolled it is conceivable that they prevent themselves from apoptosis (Reed 1999), developing senescence. Interestingly, Tao et al. 2016 showed that malign lung cancer cells treated with palbociclib alone did not change survival fraction in a signi cant manner. This should lead to more awareness using palbociclib and irradiation together in treating malign cells in vitro. Noticeably, we detected almost no supra-additive effect normalizing the combination treatment to a 2-Gy-irradiation alone. Nevertheless, it should also be mentioned that additive effects could lead to side effects in normal tissue during anti-cancer therapy.
Keeping palbociclib in mind as a disruption of the cell cycle by binding to CDK4/6 we analyzed of cell cycle distribution. Combining palbociclib with irradiation showed no response in healthy controls but in seven out of eight malign cells. The cell population in G0/G1 phase was higher in broblasts then in cancer cells and changes in G0/G1 correlate contrary with changes in G2/M. In ve out of eigth malign cells we were able to detect a decrease of cells in G2/M comparing IR and IR + inhibitor (p ≤ 0.05). In general, G2 and mitosis are most sensitive for radiation, G1 is less and S phase least radiosensitive (Sinclair 1968, Pawlik & Keyomarsi 2004. Fewer cells in the G2/M phase leads to a lower radiation sensitivity and could be an explanation for a reduced response of malignant cells to IR during palbociclib treatment. Interestingly, Xie et al. 2019 found a contrary effect compared to our data. They showed an increase of G2/M phase and as a result more apoptosis, not in melanoma cells, but in nasopharyngeal carcinoma cells. Other ndings using intracranial atypical teratoid rhabdoid tumor cell lines showed an inhibition of DNA double-strand break repair and promoted increased tumor cell apoptosis (Hashizume et al. 2016). However, it is noticeable that both groups worked with established and therefore more arti cial cell lines. For our experiments, we used skin cancer cells, generated of human skin biopsies and harboring a more primary cell status. This radioprotective effect of palbociclib in cancer cells is probably based on the accumulation of cells in the less radiosensitive G0/G1 cell cycle phase. We found that palbociclib decreases the amount of malign cell in G2/M phase signi cantly. Cancer cells are more forced to cell cycle to progress as mentioned in the "hallmarks of cancer" (Hanahan & Weinberg 2000).
Additionally, one knows that melanoma show a higher CDK4 activity because of p16 INK

Conclusion
Data of cell death (2D) analysis in healthy broblasts showed a slight tendency of decreased apoptosis and necrosis using palbociclib and irradiation simultaneously compared to IR alone. In contrast, four out of eight malign cell lines increased cell death at the same condition. Nevertheless, all cell lines especially the six representing different melanoma patients respond quiet divers. However, we were able to determine signi cant differences in our newly established 3D model showing radiosensitizing effects of palbociclib in broblasts and radioprotective effects in malign skin cancer cells. This radioprotective effect of palbociclib in cancer cells is probably based on the accumulation of cells in the less radiosensitive G0/G1 cell cycle phase as mentioned before.
Further colony forming assays implied a higher susceptibility of broblasts representing healthy tissue to the combination treatment because of minimal survival fractions. However, plotted curves indicate a radioprotective effect in broblasts and radiosensitizing effect in cancer cells. Our cell death and clonogenic experiments suggests that healthy tissue broblasts are triggered more to die via apoptosis and necrosis after treating them with kinase inhibitor and irradiation. In contrast, malign cells showed reduced survival fractions after combination therapy in our colony forming assays, which could hint for an increase of senescence.
Finally we studied weather palbociclib is able to in uence migration of cells or not, since this KI is approved in a later, progressed or metastasized stage of cancer (FDA approval 2015, Ref ID 4217399).
Our data suggests that palbociclib decelerate migration behavior of malign and non-malign cells, even in cell lines with low E-cadherin expression like MDA-MB-231 and non-malign SBLF9 cells. Advanced in vivo experiments could help to verify this observation more precisely.
All in vitro data presented in this study could give a hint for assuming palbociclib can act as a radiosensitizer causing cell death and more likely senescence, not just in HCC and HNSCC cell, but in malign melanoma cells too. Palbociclib appears to reduce the effect of ionizing radiation in vitro. Therefore, further studies are needed to assess whether a combined therapy of palbociclib and radiation is appropriate. Advanced in vivo research and highly monitored clinical trials could help to investigate how palbociclib is able to interact with ionizing radiation in healthy and tumor tissue. Availability of data and material All data generated or analyzed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.