Mitosis in Cancer Cell Increases Immune Resistance Via High Expression of HLA-G and PD-L1

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Article Mitosis in Cancer Cell Increases Immune Resistance via High Expression of HLA-G and PD-L1

Matti Ullah , Warda Aoudjeghout , Cynthia Pimpie, Marc Pocard and Massoud Mirshahi *

CAP-Paris Tech., INSERM U1275, Hôpital Lariboisière, 2, rue Ambroise-Paré, 75010 Paris, France; [email protected] (M.U.); [email protected] (W.A.); [email protected] (C.P.); [email protected] (M.P.) * Correspondence: [email protected]; Tel.: +33-1-53216765; Fax: +33-1-57216739

Received: 31 August 2020; Accepted: 14 September 2020; Published: 18 September 2020

Simple Summary: Cancer results from “aggressive division” of cells, that is unchecked by the immune cells. Cancer cells express several proteins to evade the immune response. One of the main treatment against cancer is to target and block their cell division. We found that, the expression of these proteins is increased during the mitosis stage of cell division. Therefore the blockade of cell cycle in the mitotic phase may results in increased immune resistance that can further help the cancer cells in evading immune attack.

Abstract: Cancer is a result of “aggressive” division and uncontrolled proliferation of the abnormal cells that survive attack by immune cells. We investigated the expression of HLA-G and PD-L1 with the diﬀerent stages of cancer cell division along with their role in the interaction of immune cells in vitro. Ovarian cancer (OVCAR-3) and chronic myeloid leukemia cell line (K-562) are used for this study. The correlation of protein expression with percentage of cells in each phase (G1, S and G2 phase) was evaluated through FACS. Cells were synchronized in G1, G2 and mitotic phase to evaluate gene (RT-qPCR) and protein expression (FACS). Real-time immune cell attack (RTICA) analysis with PBMCs (peripheral blood mono-nuclear cells) and cancer cells were performed. We found that cells expressing higher levels of HLA-G and PD-L1 are mainly in G2 phase and those expressing lower levels are mainly in G1 phase. Evidently, the higher expression of the two proteins was observed when synchronized in mitotic phase as compared to low expression when synchronized in G1 phase. RTICA analysis showed the presence of HLA-G delayed the lysis of the cells. In conclusion, the cancer cell can escape from immune cells in division stage that suggests the impact of mitosis index for cancer immunotherapy.

Keywords: mitosis; immune resistance; cell cycle; HLA-G; PD-L1

1. Introduction Cancer is a result of “aggressive” division and uncontrolled proliferation of abnormal cells. Ideally, the ﬁrst step in the treatment of cancer is to stop the proliferation of these cells. The classical approach against cancer is to disrupt the cell division cycle disabling the proliferation of cells. Several strategies have been used over recent decades to arrest cancer cell proliferation. Mitotic or microtubule inhibitors (like 5-ﬂuorouracil, vincristine and paclitaxel) have kept a prime place in the therapeutic approach against cancer [1–3]. However, with a better understanding of the cell cycle, new approaches have been applied in targeting other phases of the cell cycle. The progression of the cell cycle is a highly regulated step, which is controlled by various cyclin-dependent kinases such as four members of CDK family (1, 2, 4 and 6), and is known to regulate the cell cycle. The discovery of CDKs and their role in cell division cycle enlightened new approaches to target cancer division. Each of the CDK

Cancers 2020, 12, 2661; doi:10.3390/cancers12092661 www.mdpi.com/journal/cancers Cancers 2020, 12, 2661 2 of 13 expressions regulate specific cell cycle phases e.g., CDK4/6 governs the progression of cell cycle through G1 phase, CDK2 controls the progression through S-phase while CDK-1 facilitates the cells at G2/M phase to enter mitosis. Targeting these cyclin molecules has led to another therapeutic strategy in blocking the cell cycle. There are up to 10 CDK inhibitor compounds currently undergoing clinical trials. Among these compounds, CDK4/6 inhibitors are of prime importance; two CDK4/6 inhibitors have already been approved by FDA to be used as combination therapy [4,5]. Following the FDA approval of several CDK4/6 inhibitors like abemaciclib and ribociclin, initial clinical trial results have pointed out a cross-talk between cell cycle arrest and immune cell interaction [6–8]. Cyclin D-CDK4 complex regulates the PD-L1 expression through proteasome-mediated degradation by Cullin 3SPOP E3 ligase [9]. It has been reported that selective CDK4/6 pharmacologically enhances the tumor infiltration by immune cells [10,11]. Further, the use of anti-PD-L1 therapy improved the overall effect of CDK4/6 inhibitors [12]. Cancer cells exhibit one or several immune checkpoints to evade against immunity. Among several immune checkpoints, CTLA-4 and PD-L1 are the widely studied ones. The PD-L1 is expressed by the tumor cells as well as immune cells such as antigen presenting cells (APCs) and tumor-associated macrophages (TAMs) while CTLA-4 is mainly expressed by activated T-cells such as memory T-cells and regulatory T-cells [13–15]. Another protein, HLA-G, has been backed by strong evidence indicating its role in immune suppression [16–19]. Recently, we have reported that patients with ovarian carcinomatosis express HLA-G that is linked to an increase in numbers of circulating regulatory T-cells and a decrease in number of CD8 cytotoxic T-cells and NK-cells. Moreover, we found that HLA-G is expressed by different cell lines and IL-1β can induce the expression of HLA-G via NF-κB pathway [20]. As PD-L1 and HLA-G, both can be expressed by tumor cells, it would be interesting to evaluate how different phases of cell cycle affect the expression of these two proteins. In this study, we evaluated the expression of the two immune checkpoints in different cell cycle phases and the effect of their presence on interaction of immune cells and cancer cells.

2. Material and Methods

2.1. Cell Lines We chose two diﬀerent cell lines, based on the expression of the two proteins to be studied i.e., HLA-G and PD-L1. An adherent human ovarian cancer cell line (OVCAR-3) and suspension human chronic myeloid leukemia cell line (K-562), both purchased from ATCC, were used. Cell lines were cultured using RPMI-1640 medium (OVCAR-3) and IMDM medium (K-562) supplemented with 10% fetal calf serum, 50 U/mL penicillin-streptomycin and 2 mM L-glutamine in an incubator with a humidiﬁed atmosphere at 37 ◦C containing 5% CO2.

2.2. Immunofluorescence OVCAR-3 cells were cultured at 50% confluence in Lab-Tek® 3-well chamber slide for 24 h before fixation for 10 min with 4% paraformaldehyde. The cells were then washed with PBS. Non-specific binding sites were blocked using 1% BSA in PBS-tween (0.1%). Cells were then incubated with primary antibodies, anti-HLA-G (rabbit purchased from Epigentek, Farmingdale, NY, USA) and anti-PD-L1 (mouse purchased from Proteintech, Rosemont, IL, USA), respectively, for 3 h. Then, cells were washed with PBS-Tween (0.1%) and incubated with secondary antibodies (anti-rabbit AF488 and anti-mouse AF594; Invitrogen, Waltham, MA, USA) for 1 h. The incubations were performed at room temperature with mild agitation. The slides were then washed and mounted with DAPI containing aqueous mounting medium by Vectashield® for images to be taken using ZEISS LSM 900 confocal microscope. Cancers 2020, 12, 2661 3 of 13

2.3. FACS Analysis for Cell Cycle and Protein Expression

2.3.1. FACS For FACS analysis, cells were stained using an indirect staining method. Cells were collected from the flasks (with or without treatment), and were fixed using ice-chilled 70% ethanol at 4 ◦C. The cells were then washed and incubated with primary antibodies (as mono-staining) overnight at 4 ◦C at a concentration of 1 µg for HLA-G (Epigentek, USA) and 1.25 µg for PD-L1 (Proteintech, USA) per 100,000 cells. In parallel, the cells were stained with equivalent amount of isotype antibodies (Figure S4) i.e., rabbit polyclonal IgG and mouse monoclone IgG1 antibody (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA). On the following day, the cells were then washed and incubated with secondary antibodies (anti-rabbit AF488 and anti-mouse AF546; Invitrogen) for 2 h. To stain the cells in mitosis, we used MPM2 antibody (Anti-phospho-Ser/Thr-Pro, Cy5 conjugate; Sigma-Aldrich, Saint Quentin Fallavier, France) at concentration of 2 µg per 100,000 cells. The cells were analyzed using 3-Laser Flow cytometer “Gallios” by Beckmann Coulter and the results were analyzed using “Kaluza Analysis 2.1” software. Cell cycle was recorded through DAPI, using Michel H. Fox algorithm (built-in Software).

2.3.2. FACS Analysis For MFI (mean fluorescence intensity), mode fluorescence intensity was evaluated as it expressed the fluorescence of the majority of the cells. Further, for pre-screening, the fluorescence peak was gated in ten equal parts termed as deciles (marked as A to J with 10% of cells in each gate) in increasing intensities of fluorescence (Figure S2), so that cells in 1st decile expressed the least intensity of the proteins and 10th decile represented cells that expressed the highest protein levels. The cell cycle using DAPI was determined for each decile, and correlation was evaluated for protein level using mean fluorescence intensity of each gated region (Figure S2).

2.4. Cell Synchronization Cell lines were synchronized in G1 phase, G2 phase and Mitotic phase using lovastatin [21], CDK-1 inhibitor (RO-33022 by Sigma Aldrich) [22] and nocodazole (mitotic inhibitor) [23]. The synchronizing agents were dissolved in DMSO, therefore DMSO-treated cells were used as control. Each of the cell lines was treated for 24 h with the agents to synchronize cells in a speciﬁc phase. The concentration (as µg/mL for 2 million cells in each condition) is given in the Table1 for each cell line.

Table 1. Concentrations (µg/mL) of synchronizing agents

Synchronising Agent Ovarian Cancer (OVCAR-3) Chronic Myeloid Leukemia Cell Line (K-562) Lovastatin 32 27 CDK-1 9 5 Nocodazole 0.48 0.4

2.5. mRNA Expression of HLA-G and PD-L1 via qPCR RNA was isolated from the cells using ReliaPrep™ RNA Miniprep Systems by Promega following the treatment with cell cycle inhibitors. The RNA was reverse-transcripted to cDNA using Maxima First Strand cDNA Synthesis Kit. The cDNA was then used at a concentration of 2.5 ng/10 uL reaction in LightCycler 96 by Roche using SYBR green master mix in 3-step ampliﬁcation. The relative expression was measured through ∆∆CT method using beta-actin as housekeeping gene. The primer sequences were: beta-actin (sens: AGA GCT ACG AGC TGC CTG AC); anti-sense: AGC ACT GTG TTG GCG TAC AG), HLA-G (sens: GCG GCT ACT ACA ACC AGA GC; anti-sense: GAG GTA ATC CTT GCC ATC GTA G), PD-L1 (sens: CAA AGA ATT TTG GTT GTG GA; anti-sense: AGC TTC TCC TCT CTC TTG GA) [24,25]. Cancers 2020, 12, 2661 4 of 13

2.6. Real-Time Immune Cell Attack (RTICA) Analysis In order to determine the eﬀect of HLA-G and PD-L1 on interaction of immune cells with cancer Cancers 2020, 12, x 4 of 12 cells, we co-cultured OVCAR-3 (stained using NucBlue Live ReadyProbes™, ThermoFisher Scientiﬁc) withused PBMCscells synchronized in 5:1 ratio in RPMIG1 phase medium (lovastatin) with or and without mitotic HLA-G phase (3(nocodazole). ng/mL) and The PD-L1 images (3 ng /weremL). Weanalyzed performed using cinematographyImage-J. The cell bywas taking considered pictures lysed at intervalas soon ofas 30cytoplasm s. The PBMCsstarted leaking were collection and the immunefrom human cells blood were followingconsidered Ficoll attached separation if their protocol attachment (oral remained consent taken). constant In parallel,for the 10 we frames usedcells i.e., 5synchronized min (supplementary in G1 phase video (lovastatin) provided; and Video mitotic S1; phase for unsynchronized (nocodazole). The cells images in normal were analyzed culture medium).using Image-J. The cell was considered lysed as soon as cytoplasm started leaking and the immune cells were considered attached if their attachment remained constant for the 10 frames i.e., 5 min (Supplementary2.7. Statistical Analysis Video provided; Video S1; for unsynchronized cells in normal culture medium).

2.7. StatisticalThe statistical Analysis analysis was performed using GraphPad Prism 6. One-way ANOVA (post-Hoc Tukey’s test) was performed to compare the difference between the two groups, while correlation The statistical analysis was performed using GraphPad Prism 6. One-way ANOVA (post-Hoc was calculated as spearman correlation. Tukey’s test) was performed to compare the difference between the two groups, while correlation was 3.calculated Results as spearman correlation. 3. Results 3.1. HLA-G and PD-L1 Expression in Dividing and Non-Dividing Cells 3.1. HLA-GThe immunofluorescence and PD-L1 Expression for in HLA-G Dividing and and PD-L1 Non-Dividing in OVCAR-3 Cells cells showed variable expression of theThe two immunofluorescence proteins in monotype for HLA-G cell line. and Some PD-L1 of in the OVCAR-3 cells expressed cells showed higher variable protein expression intensity of comparedthe two proteins to other in monotypecells. We found cell line. that Some the ofdividi theng cells cells expressed expressed higher higher protein protein intensity expression compared for HLA-Gto other as cells. well We as found PD-L1 that compared the dividing to non-dividing cells expressed cells higher (Figure protein 1), and expression this increase for HLA-G in expression as well variesas PD-L1 through compared different to non-dividing mitotic stages cells (Figure (Figure S1).1 We), and found this the increase high HLA-G in expression expression varies among through the cellsdifferent in prophase, mitotic stages which (Figure is the S1).starting We found point theof cell high division. HLA-G This expression expression among remains the cells high in prophase,until the whichtelophase. is the However, starting pointthe expression of cell division. of HLA-G This decreases expression in remains cytokinesis, high untilalthough thetelophase. it still remains However, high asthe compared expression to ofnon-dividing HLA-G decreases cells. This in cytokinesis, suggests that although the HLA-G it still expression remains high is increased as compared during to mitosisnon-dividing but starts cells. to This decrease suggests again that at the the HLA-G end of expressionthe mitosis. is increasedFurther, we during found mitosis that the but PD-L1 starts expressionto decrease was again high at thethroughout end of the the mitosis. mitotic Further, stages as we well found as thatin cytokinesis the PD-L1 as expression compared was to non- high dividingthroughout cells. the mitotic stages as well as in cytokinesis as compared to non-dividing cells.

Figure 1. ImmunofluorescenceImmunoﬂuorescence for for HLA-G HLA-G and and PD-L1 inin OVCAR-3OVCAR-3 cells.cells. OVCAR-3 OVCAR-3 cells cells were cultured in cell chamber and stained stained for for HLA-G HLA-G (gr (green)een) and and PD-L1 PD-L1 (red) (red) and and DA DAPIPI (blue) (blue) for for nucleus. nucleus. An increased expression of the two proteins (HLA-G and PD-L1) was observed in dividing cells (thick white arrows arrows with with dividing dividing nuclear nuclear material) material) compared compared to non-dividing to non-dividing cells cells(thin (thinwhite white arrows; arrows; scale µ bar:scale 10 bar: µm). 10 m).

3.2. Pre-Screening through FACS The cells (using expression intensity peak) were gated in deciles with increasing fluorescence expression (Figure 2D, Supplementary Figure S2). The cell cycle was analyzed for each gated cell to find out the correlation between cell cycle phase and expression intensity. We found the increasing percentage of cells in G2 while there was a decreasing percentage of cells in G0/G1 phase from 1st decile (low expression) to 10th decile (high expression) for HLA-G in both the cell lines (Figure 2A).

Cancers 2020, 12, 2661 5 of 13

3.2. Pre-Screening through FACS The cells (using expression intensity peak) were gated in deciles with increasing ﬂuorescence expression (Figure2D, Supplementary Figure S2). The cell cycle was analyzed for each gated cell to

Cancersﬁnd out 2020 the, 12 correlation, x between cell cycle phase and expression intensity. We found the increasing5 of 12 percentage of cells in G2 while there was a decreasing percentage of cells in G0/G1 phase from 1st

Thedecile expression (low expression) intensity to of 10th HLA-G decile correlated (high expression) positively for with HLA-G percentage in both of the cells cell in lines G2 (r (Figures = 0.932 A).for TheK-562 expression and rs = 0.94 intensity for OVCAR-3) of HLA-G while correlated there was positively a negative with correlation percentage with of cells the inpercentage G2 (rs = 0.93of cells for inK-562 G0/G1 and phase rs = 0.94 (rs = for −0.91 OVCAR-3) for K-562 while and r theres = −0.93 was for a negativeOVCAR-3). correlation This shows with that the majority percentage of cells of cells in lowin G0 or/G1 intermediate phase (rs = expression0.91 for K-562 of HLA-G and rs were= 0.93 present for OVCAR-3). in G0/G1 phase This shows while thatthe majority majority of cells − − thatin low show or intermediate high expression expression of HLA-G of HLA-Gwere present were presentin G2 phase in G0 (Figure/G1 phase 2C). while There the was majority no significant of cells differencethat show highfound expression between percentage of HLA-G of were S-phase present cells in from G2 phase 1st decile (Figure to 210thC). Theredecile wasand no signiﬁcantcorrelation wasdiﬀerence observed found for between percentage percentage of S-phase of S-phasewith expression cells from of 1stHLA-G. decile to 10th decile and no correlation was observed for percentage of S-phase with expression of HLA-G.

Figure 2. Cell cycle phases based on expression of HLA-G and PD-L1 (FACS). OVCAR-3 and K-562 cells were stained for HLA-G (a) and PD-L1 (b). Cell cycle analyzed after gating protein expression Figure 2. Cell cycle phases based on expression of HLA-G and PD-L1 (FACS). OVCAR-3 and K-562 peak in 10 equal parts (deciles; A-J) in increasing protein expression. (A). For HLA-G, both the cell lines cells were stained for HLA-G (a) and PD-L1 (b). Cell cycle analyzed after gating protein expression showed that the cells present in first deciles (low expression) were mainly in G0/G1 phase, while those peak in 10 equal parts (deciles; A-J) in increasing protein expression. (A). For HLA-G, both the cell in higher deciles have the majority of the cells in G2 phase. (B). For PD-L1, The G0/G1 and S-phase lines showed that the cells present in first deciles (low expression) were mainly in G0/G1 phase, while cells showed similar percentage of cells present in each phase among different gates (from low to high those in higher deciles have the majority of the cells in G2 phase. (B). For PD-L1, The G0/G1 and S- expression). The G2 phase cells were present more in higher intensity gates compared to lower ones. phase cells showed similar percentage of cells present in each phase among different gates (from low (C). The FACS analysis showed strong positive correlation of G2 phase (rs > 0.9) with HLA-G and to high expression). The G2 phase cells were present more in higher intensity gates compared to lower PD-L1 in both the cell lines, while strong negative correlation of G0/G1 phase was found only for ones. (C). The FACS analysis showed strong positive correlation of G2 phase (rs > 0.9) with HLA-G HLA-G (rs > 0.9) but not with PD-L1. The S-phase did not show any correlation for HLA-G with either and PD-L1 in both the cell lines, while strong negative correlation of G0/G1 phase was found only for of the cell lines, and weak negative correlation (rs = 0.3) was found for PD-L1. (D). An image of the HLA-G (rs > 0.9) but not with PD-L1. The S-phase did− not show any correlation for HLA-G with either peak expression gated for 10 deciles marked as A to J with increasing expression of the protein. of the cell lines, and weak negative correlation (rs = −0.3) was found for PD-L1. (D). An image of the peakFor PD-L1, expression we didgated not for find 10 deciles any regular marked pattern as A to forJ with G0 increasing/G1 and S-phase expression (Figure of the2 B).protein. Although the cells with lowest expression of PD-L1 (first and second deciles) consisted mostly of G0/G1 phase (near 60%).For Moreover, PD-L1, we very did low not percentage find any regular of G2 cellspattern (less for than G0/G1 20%) and were S-phase present (Figure in low 2B). expressing Although cells, the whilecells with majority lowest of expression the G2 phase of PD cells-L1 showed (first and high second expression deciles) of cons PD-L1.isted No mostly Significant of G0/G1 correlation phase (near was 60%).found Moreover, for G0/G1 very phase low with percentage expression of G2 intensity cells (les ofs PD-L1, than 20%) while were G2 present phaseshowed in low expressing strong positive cells, while majority of the G2 phase cells showed high expression of PD-L1. No Significant correlation was correlation (rs = 0.79 for K-562 and rs = 0.92 for OVCAR-3). For PD-L1, S-phase showed weak found for G0/G1 phase with expression intensity of PD-L1, while G2 phase showed strong positive negative correlation (rs = 0.36 for K-562 and rs = 0.45 for OVCAR-3) with PD-L1 expression intensity − − (Figurecorrelation2C). (rs = 0.79 for K-562 and rs = 0.92 for OVCAR-3). For PD-L1, S-phase showed weak negative correlation (rs = −0.36 for K-562 and rs = −0.45 for OVCAR-3) with PD-L1 expression intensity (Figure 2C).

Cancers 2020, 12, 2661 6 of 13

3.3. Cell Synchronization The cells were treated with lovastatin, CDK-1 inhibitor and nocodazole to synchronize in G1, G2 phase and mitotic phase, respectively. DMSO-treated cells were used as control unsynchronized cells. Figure3A shows the percentage of cells in three di ﬀerent phases after treatment. For OVCAR-3 cells treated with nocodazole, only non-adherent cells were collected to further enrich mitotic cells. Further, we performed FACS analysis using MPM2 staining (Figure3B) in order to determine the mitotic index Cancersfor 2020 CDK-1, 12, x inhibitor- and nocodazole-treated cells. We found that CDK-1 inhibitor-treated cells have6 of 12 very low mitotic index (Figure3B) with percentage of MPM2 positive cells (4.6 0.6) compared to ± 0.6) comparedthose treated to withthose nocodazole treated with (87.8 nocodazole1.3). Therefore, (87.8 CDK-1 ± 1.3). inhibitor-treated Therefore, CDK-1 cells inhibitor-treated were majorly in G2 cells ± werephase majorly while in nocodazoleG2 phase while blocked nocodazole cells in mitotic blocked phase. cells in mitotic phase.

Figure 3. Cell phase following cell cycle inhibition. The two cell lines were synchronized in G1 phase Figureusing 3. Cell lovastatin, phase G2following phase using cell CDK-1cycle inhibition. inhibitor and The mitosis two cell phase lines using were nocodazole. synchronized (A). Percentage in G1 phase usingof lovastatin, each cells in G2 diﬀ erentphase cell using cycle phasesCDK-1 (K-562, inhibitor right and OVCAR-3mitosis phase left) analyzed using throughnocodazole. FACS (A). Percentage(Michael of H. each Fox algorithm) cells in usingdifferent DAPI cell is presented. cycle phases (B). Percentage (K-562, ofright positive and cells OVCAR-3 with MPM2 left) staining analyzed throughshow FACS mitotic (Michael cells (OVCAR-3) H. Fox algori followingthm) using CDK-1 DAPI inhibitor is presented. and nocodazole (B). Percentage treatment. of Nocodazole positive cells with blocksMPM2 cells staining in mitotic show phase mitotic while low cells expression (OVCAR-3) of MPM2 following in CDK-1 CDK-1 inhibitor-treated inhibitor cells and shows nocodazole that treatment.it blocks Nocodazole cells only in G2blocks phase ce (****,lls inp < mitotic0.0001unpaired phase whilet-test). low expression of MPM2 in CDK-1 3.4.inhibitor-treated HLA-G (Protein cells and shows Gene) that Expression it blocks in cells Synchronized only in G2 Cells phase (****, p < 0.0001 unpaired t-test).

3.4. HLA-GBoth (Protein the cell and lines Gene) when Expression synchronized in Synchronized in different Cells phases, showed difference in the HLA-G expression (protein as well as mRNA expression). In K-562 cells (Figure4A) we found that the lovastatin-treatedBoth the cell lines cells when showed synchronized lowest expression in different of the HLA-G phases, protein showed compared difference to CDK-1 in inhibitor-the HLA-G expression(p < 0.005) (protein and nocodazole-treated as well as mRNA cells expression). (p < 0.0001). In However, K-562 cells there (Figure was no significant4A) we found difference that the lovastatin-treatedin expression compared cells showed to unsynchronized lowest expres cells.sion Further, of the the nocodazole-treatedHLA-G protein compared cells showed to even CDK-1 inhibitor-higher ( expressionp < 0.005) comparedand nocodazole-treated to CDK-1 inhibitor-treated cells (p cells< 0.0001). (p < 0.005). However, However, there the was gene expressionno significant differencefor HLA-G in expression in K-562 cell compared lines (Figure to4B) unsynchron was only increasedized cells. in CDK-1 Further, inhibitor-treated the nocodazole-treated cells ( p < 0.0001 cells showedcompared even higher to lovastatin expression and nocodazole. compared There to CDK-1 was no inhibitor-treated significant difference cells found (p < for 0.005). gene expressionHowever, the of HLA-G between unsynchronized cells, lovastatin-treated cells and nocodazole-treated cells. gene expression for HLA-G in K-562 cell lines (Figure 4B) was only increased in CDK-1 inhibitor- treated cells (p < 0.0001 compared to lovastatin and nocodazole. There was no significant difference found for gene expression of HLA-G between unsynchronized cells, lovastatin-treated cells and nocodazole-treated cells.

Cancers 2020, 12, 2661 7 of 13 Cancers 2020, 12, x 7 of 12

Figure 4.4. HLA-G (protein andand mRNA)mRNA) expressionexpression byby K-562K-562 andand OVCAR-3.OVCAR-3. (A).). MeanMean fluorescencefluorescence intensityintensity (MFI)(MFI) usingusing modemode fluorescencefluorescence intensityintensity isis shownshown forfor thethe twotwo cellcell lines.lines. DMSO-treatedDMSO-treated cellscells are shownshown as as a controla control with with unsynchronized unsynchronized cells. Nocodazole-treatedcells. Nocodazole-treated cells have cells the highesthave the expression highest ofexpression HLA-G protein of HLA-G in each protein of the in celleach lines of the while cell lovastatin-treatedlines while lovastatin-treated cells showed cells the showed lowest expression, the lowest notexpression, different tonot unsynchronized different to cells.unsynchronized The CDK-1 inhibitor-treatedcells. The CDK-1 cells inhibitor-treated showed intermediate cells expression showed forintermediate K-562 cells expression while for OVCAR-3 for K-562 cells, cells expression while for wasOVCAR-3 higher thancells, lovastatin-treated expression was cellshigher but than not dilovastatin-treatedfferent than nocodazole-treated cells but not differe cells.nt (thanB). qPCR nocodazole-treated analysis showed cells. that (B both). qPCR cell analysis lines express showed higher that geneboth expressioncell lines express when treatedhigher withgene CDK-1 expression inhibitor when (G2 treated phase) with compared CDK-1 to inhibitor lovastatin (G2 (G1) phase) and nocodazole (mitotic)-treated cells. No difference was observed for gene expression in lovastatin compared to lovastatin (G1) and nocodazole (mitotic)-treated cells. No difference was observed for (G1)-treated cells compared to nocodazole (mitotic)-treated cells in K-562 cell line. However, OVCAR-3 gene expression in lovastatin (G1)-treated cells compared to nocodazole (mitotic)-treated cells in K- cells showed the lowest mRNA expression in nocodazole-treated cells that was significantly lower than 562 cell line. However, OVCAR-3 cells showed the lowest mRNA expression in nocodazole-treated lovastatin-treated cells (One-Way ANOVA; Tukey’s test * < 0.05, ** < 0.005, *** < 0.001, **** < 0.0001). cells that was significantly lower than lovastatin-treated cells (One-Way ANOVA; Tukey’s test * < Similar0.05, ** < results0.005, *** were < 0.001, observed **** < 0.0001). for OVCAR-3 cell lines (Figure4A). The lovastatin-treated cells showed lowest expression compared to CDK-1 inhibitor- (p < 0.005) and nocodazole (p < 0.001)-treated Similar results were observed for OVCAR-3 cell lines (Figure 4A). The lovastatin-treated cells cells. In contrast to K-562 cell line, there was no significant difference found between CDK-1 showed lowest expression compared to CDK-1 inhibitor- (p < 0.005) and nocodazole (p < 0.001)- inhibitor-treated cells compared to nocodazole-treated cells. This shows that the HLA-G expression treated cells. In contrast to K-562 cell line, there was no significant difference found between CDK-1 in OVCAR-3 cell lines is increased during G2 phase and does not change during mitosis. Moreover, inhibitor-treated cells compared to nocodazole-treated cells. This shows that the HLA-G expression similar to K-562 cell lines, we found that the gene expression (Figure4B) was highest in CDK-1 in OVCAR-3 cell lines is increased during G2 phase and does not change during mitosis. Moreover, inhibitor-treated OVCAR-3 cells compared to lovastatin- (p < 0.005) and nocodazole (p < 0.0001)-treated similar to K-562 cell lines, we found that the gene expression (Figure 4B) was highest in CDK-1 cells. In addition, the lovastatin-treated cells showed increased gene expression of HLA-G compared inhibitor-treated OVCAR-3 cells compared to lovastatin- (p < 0.005) and nocodazole (p < 0.0001)- to nocodazole-treated cells (p < 0.05). treated cells. In addition, the lovastatin-treated cells showed increased gene expression of HLA-G 3.5.compared PD-L1 to (Protein nocodazole-treated and mRNA) Expression cells (p

3.5. PD-L1We found (Protein an increasedand mRNA) PD-L1 Expression protein in expressionSynchronized when Cells cells were treated with nocodazole for both the cell lines (Figure5A). We found a significant low protein expression in CDK-1 inhibitor-treated cells asWe compared found an toincreased nocodazole PD-L1 (p < protein0.005) inexpression both the cellwhen lines. cells The were lovastatin-treated treated with nocodazole cells showed for similarboth the expression cell lines to(Figure CDK-1 5A). inhibitor-treated We found a signif cells,icant both low significantly protein expressi lower thanon in nocodazole-treated CDK-1 inhibitor- cells.treated Interestingly, cells as compared lovastatin-treated to nocodazole OVCAR-3 (p < 0.005) cells in showed both the significantly cell lines. The lower lovastatin-treated expression of PD-L1 cells comparedshowed similar to unsynchronized expression to CDK-1 cells (DMSO), inhibitor-treate suggestingd cells, that both cells, significantly when synchronized lower than innocodazole- G1 phase, showtreated lower cells. expression Interestingly, of PD-L1. lovastatin-treated In contrast, OVCAR-3 No change cells was showed observed significantly for lovastatin-treated lower expression K-562 cellsof PD-L1 as compared compared to to unsynchronized unsynchronized cells. cells (DMSO), suggesting that cells, when synchronized in G1 phase, show lower expression of PD-L1. In contrast, No change was observed for lovastatin-treated K-562 cells as compared to unsynchronized cells.

Cancers 2020, 12, 2661 8 of 13 Cancers 2020, 12, x 8 of 12

Figure 5. PD-L1 (protein and mRNA) expression in K-562 and OVCAR-3. (A). MFI using mode Figure 5. PD-L1 (protein and mRNA) expression in K-562 and OVCAR-3. (A). MFI using mode fluorescence intensity is shown for the two cell lines. Both the cell lines showed significant increase in fluorescence intensity is shown for the two cell lines. Both the cell lines showed significant increase PD-L1 protein expression in the cells treated with nocodazole (mitotic), no difference was observed in PD-L1between protein lovastatin- expression (G1) and in CDK-1the cells inhibitor treated (G2)-treated with nocodazole cells. (B (mitotic),). Similar gene no difference expression was to HLA-G observed betweenwas foundlovastatin- for PD-L1 (G1) where and CDK-1 G2 phase inhibitor cells had (G2)-treated higher gene expressioncells. (B). Similar compared gene to cellsexpression treated withto HLA- G waslovastatin found for (G1) PD-L1 and nocodazole where G2 (mitotic), phase cells while had there high waser no gene diff erenceexpression found compared in the gene to expression cells treated withbetween lovastatin cells (G1) treated and with nocodazole lovastatin (G1)(mitotic), and nocodazole while there (Mitotic). was (One-Wayno difference ANOVA; found Tukey’s in the test gene expression* < 0.05, between ** < 0.005, cells *** < treated0.001, **** with< 0.0001).lovastatin (G1) and nocodazole (Mitotic). (One-Way ANOVA; Tukey’s test * < 0.05, ** < 0.005, *** < 0.001, **** < 0.0001). We found the highest gene expression of PD-L1 in CDK-1 inhibitor-treated cells for both of the cell lines (Figure5B). Lovastatin and nocodazole showed significantly lower gene expression of PD-L1 in We found the highest gene expression of PD-L1 in CDK-1 inhibitor-treated cells for both of the both the cell lines when compared to CDK-1 inhibitor-treated cells (p < 0.0001 for K-562 and p < 0.005 for cell lines (Figure 5B). Lovastatin and nocodazole showed significantly lower gene expression of PD- OVCAR-3). There was no significant difference found between lovastatin- and nocodazole-treated cells. L1 in both the cell lines when compared to CDK-1 inhibitor-treated cells (p < 0.0001 for K-562 and p < 0.0053.6. for HLA-G OVCAR-3). and Mitotic There Blockade was no Suppress significant the Immune difference Cell Attack found between lovastatin- and nocodazole- treated cells.When we cultured OVCAR-3 cells with PBMCs in presence of HLA-G and PD-L1, we found that presence of PD-L1 initially resulted in increased lysis of OVCAR-3 cells. While the presence of HLA-G 3.6. HLA-Gdelayed and the Mitotic lysis of Blockade the cells (FigureSuppress6A). the The Immune statistical Cell analysisAttack showed that HLA-G significantly increasesWhen we the cultured time required OVCAR-3 for thecells lysis with of PBMCs half of thein presence cancer cells of HLA-G by 30 min and while PD-L1, there we was found no that difference found in the presence of PD-L1 compared to regular culture medium (RPMI; Figure6B). presence of PD-L1 initially resulted in increased lysis of OVCAR-3 cells. While the presence of HLA- Hence, presence of HLA-G in-vitro, provided a better immune protective effect than PD-L1 to the G delayed the lysis of the cells (Figure 6A). The statistical analysis showed that HLA-G significantly cancer cells. Moreover, when we analyzed the interaction of the immune cells, there was less interaction increasesfound the between time immunerequired cells for andthe cancerlysis of cells half in of presence the cancer of HLA-G cells asby well 30 min as PD-L1 while (Figure there6 C).was no differenceAdditionally, found thisin the decreased presence interaction of PD-L1 between compared cancer tocells regular and immuneculture medium cells was found(RPMI; when Figure we 6B). Hence,cultured presence cells of synchronized HLA-G in-vitro, in mitotic provided phase (Figure a better6D; immune Figure S3). protective However, effect the interaction than PD-L1 with to the cancerlovastatin-treated cells. Moreover, cells when (G1) was we significantly analyzed higherthe in comparedteraction toof nocodazole-treated the immune cells, cells there (mitosis). was less interaction found between immune cells and cancer cells in presence of HLA-G as well as PD-L1 (Figure 6C). Additionally, this decreased interaction between cancer cells and immune cells was found when we cultured cells synchronized in mitotic phase (Figure 6D; Figure S3). However, the interaction with lovastatin-treated cells (G1) was significantly higher compared to nocodazole- treated cells (mitosis).

Cancers 2020, 12, 2661 9 of 13 Cancers 2020, 12, x 9 of 12

FigureFigure 6. Co-culture 6. Co-culture of OVCAR-3 of OVCAR-3 cells cells with with PBMCs. PBMCs. (A (A). ).Time-dependent Time-dependent lysis ofof OVCAR-3OVCAR-3 cells cells in RPMIin medium RPMI medium compared compared to presence to presence of HLA-G of HLA-G and andPD-L1. PD-L1. PD-L1 PD-L1 initially initially increases increases the the lysis, lysis, while while HLA-G resulted in delayed lysis of the cancer cells compared to OVCAR-3 cultured in absence of HLA-G resulted in delayed lysis of the cancer cells compared to OVCAR-3 cultured in absence of two two proteins. (B). HLA-G increased the time required for the lysis of half of the population, but no proteins. (B). HLA-G increased the time required for the lysis of half of the population, but no difference with PD-L1. (C). PD-L1 and HLA-G, both decreased the attachment of the immune cells as a differencedecrease with in totalPD-L1. number (C). ofPD-L1 cells attached and HLA-G, to the cancerboth decreased cells. (D). Wethe found attachment that the of interaction the immune between cells as a decreaseimmune in cells total and number mitotic cellsof cell (nocodazole)s attached wasto the significantly cancer cells. low compared(D). We tofound G1-synchronized that the interaction cells between(lovastatin) immune or unsynchronizedcells and mitotic cells. cells (nocodazole) was significantly low compared to G1- synchronized cells (lovastatin) or unsynchronized cells. 4. Discussion 4. DiscussionCancer cells express PD-L1 and other immune checkpoints to protect themselves against the immune system. Among these, the PD-1/PD-L1 pathway is one of the most widely studied immune Cancer cells express PD-L1 and other immune checkpoints to protect themselves against the checkpoints expressed by cancer cells. Another newly recognized immune checkpoint is HLA-G, immunewhich system. was initially Among discovered these, the at maternal–fetalPD-1/PD-L1 path interfaceway withis one a functionof the most to protect widely the studied fetus against immune checkpointsmaternal expressed immune system. by cancer CDK 4cells./6 inhibitors Another that ne blockwly cells recognized in G1 phase immune have been checkpoint shown to enhance is HLA-G, whichimmune was initially cells’ infiltration discovered of tumorsat maternal–fetal [11]. Therefore, inte itrface would with be importanta function to to study protect the expressionthe fetus against of maternalPD-L1 immune as well as system. HLA-G inCDK different 4/6 cellinhibitors cycle phases that toblock evaluate cells how in theG1 cell phase division have cycle been affects shown the to enhanceexpression immune of these cells’ two infiltration immune checkpoints. of tumors We[11]. used Therefore, two different it would cell lines be i.e., important OVCAR-3 to which study is the expressionadherent of humanPD-L1 ovarian as well cancer as HLA-G cell line in and different K-562 humancell cycle leukemia phases suspension to evaluate cell how line. Thethe cell linesdivision cyclewere affects chosen the basedexpression on the of expression these two of theimmune two proteins checkpoints. i.e., HLA-G We andused PD-L1. two different cell lines i.e., OVCAR-3We which found is the adherent difference human in expression ovarian of cancer the two cell proteins line and (HLA-G K-562 and human PD-L1) leukemia among the suspension cells of the monoclonal cell line, OVCAR-3. Immunofluorescence analysis showed that the cells in mitotic phase cell line. The cell lines were chosen based on the expression of the two proteins i.e., HLA-G and PD- express higher levels of HLA-G and PD-L1 compared to non-dividing cells. In addition, the expression L1. is not uniform throughout the mitosis, rather it changes through different mitotic stages, with HLA-G expressionWe found decreasingthe difference at the in endexpression of the mitosis of the and two the proteins start of (HLA-G the cytokinesis and PD-L1) while PD-L1among levels the cells of theincrease monoclonal in cytokinesis cell line, as OVCAR-3. compared Immunofluoresc to mitotic phase.ence This suggestsanalysis thatshowed cancer that cells the can cells be in more mitotic phaseresistant express to higher immune levels attack of during HLA-G division and asPD-L1 compared compared to the restingto non-dividing stage. It has cells. been reportedIn addition, that the expressionPD-L1 knockoutis not uniform does not throughout affect the tumor the mitosis, growth in rather vivo, thereforeit changes the through increased different expression mitotic of PD-L1 stages, with canHLA-G be seen expression as an additional decreasing immune at the suppressive end of th mechanisme mitosis and adapted the start by the of cells, the thatcytokinesis further need while to PD- L1 levelsbe studied increase [15]. in cytokinesis as compared to mitotic phase. This suggests that cancer cells can be more resistantMoreover, to immune we performed attack FACS during analysis division (in the as two compared cell lines K-562to the and resting OVCAR-3), stage. where It has we been reportedgated that the unsynchronizedPD-L1 knockout cells does based not on affect their proteinthe tumor expression growth and in analyzedvivo, therefore the cell cyclethe increased after culturing cells in their regular culture conditions. We found that the cells express higher levels of expression of PD-L1 can be seen as an additional immune suppressive mechanism adapted by the HLA-G near or during division as the majority of cells that have higher protein levels were mainly in cells, that further need to be studied [15]. Moreover, we performed FACS analysis (in the two cell lines K-562 and OVCAR-3), where we gated the unsynchronized cells based on their protein expression and analyzed the cell cycle after culturing cells in their regular culture conditions. We found that the cells express higher levels of HLA-G near or during division as the majority of cells that have higher protein levels were mainly in G2 phase as compared to ones with low expression that were found to be in G0/G1 phase. However, the positive correlation of PD-L1 expression only increases in G2 phase. This confirms the previous

Cancers 2020, 12, 2661 10 of 13

G2 phase as compared to ones with low expression that were found to be in G0/G1 phase. However, the positive correlation of PD-L1 expression only increases in G2 phase. This confirms the previous observation conducted in OVCAR-3 through immunofluorescence in both the cell lines confirming that both HLA-G and PD-L1 expression increases near cell division. Further, to confirm these initial findings, the expression (protein as well as mRNA) was analyzed following cell synchronization in different phases of cell division. Lovastatin was used to arrest cells in G1 phase, CDK-1 inhibitor to block in G2 phase and nocodazole to block the cells in mitotic phases. DMSO-treated cells were used as control unsynchronized cells as each of the synchronizing agents was dissolved in DMSO. High expression of MPM-2 in nocodazole-treated cells compared to CDK-1 inhibitor-treated cells revealed that CDK-1 inhibitor blocks cells in G2 phase while nocodazole blocks cells in mitotic phase. We found a significant increase in protein expression of HLA-G as well as PD-L1 in cells arrested in mitotic phase confirming that mitotic cells express higher levels of the two proteins as compared to other two phases. Moreover, the OVCAR-3 cell lines showed increase in expression of HLA-G even in G2 phase. However, the expression of HLA-G in K-562 cell line was significantly lower in G2 phase compared to mitotic phase suggesting that although the cells show higher expression in mitotic phase, the increase in G2 phase is cell line dependent. We found that the cells arrested in G1 phase showed lowest expression of HLA-G compared to cells arrested in G2 phase and mitotic phase. Further, the significant lower expression of PD-L1 in OVCAR-3 for lovastatin-treated cells, compared to unsynchronized cells, suggests that cells in G1 phase show low expression of PD-L1. This may be the possible reason for CDK4/6 inhibitors enhancing anti-tumor immunity, as the cells in G1 phase show lower expression of both the immune suppressive proteins [6]. However, this decreased expression can be cancer type-dependent as we did not observe such difference in K-562 cell line. Moreover, we found that among different isoforms of HLA-G, only the sHLA-G1 isoform was expressed by the two cell lines, confirmed through intracellular MEM-G9 staining. We did not find the presence of membrane-bound HLA-G or sHLA-G5 in both of the cell lines (results not shown). Further, we found that the gene expression for each protein in both the cell lines was higher in G2 phase compared to G1 and mitotic phase. This increased transcription may be translated to higher protein expression in mitotic phase. Further, the decreased transcription in mitotic phase can be the result of a feedback mechanism, and may be responsible for decreased protein expression in G1 phase; however, this needs to be further explored. In order to see how this altered expression, affects the immune cell interaction with tumor cells, we co-cultured PBMCs with OVCAR-3 cells and filmed their interaction. This co-culture technique has been used to evaluate the inhibitory effect of cancer cells on T-cell activation and their cytokine secretion [26]. However, we developed a real-time attack assay by observing that the immune cells interaction can be evaluated through cinematography. Firstly, we performed this assay in presence of HLA-G and PD-L1 and we found that both HLA-G and PD-L1 can block the interaction of immune cells with cancer cells, however, in-vitro HLA-G provided stronger immune protection to cancer cells compared to PD-L1 that initially resulted in increased lysis of the cancer cells. Similarly, the cells synchronized in mitotic phase showed decreased interaction with immune cells compared to G1 synchronized or unsynchronized cells. The cell lysis analysis was not reliable for the synchronized cells as the release from the synchronization itself resulted in apoptosis. These results suggest that the cancer treatment that results in mitotic blockade may result in lowering the immune cells interaction with cancer cells through increased expression of HLA-G as well as PD-L1. Although, the increased expression of PD-L1 in mitosis has already been reported [9], our study suggests that use of the mitotic inhibitor cells may result in increased immune resistance in cancer cells. The cells that do not undergo apoptosis during mitotic inhibition therapy may not be susceptible to attack by the immune system, hence resulting in cancer relapse. Further, we can explain the anti-tumor response of anti-PD-L1 with the use of CDK4/6 inhibitors owing to decreased expression of PD-L1 in G1 phase (that synergizes with anti-PD-L1 therapy). However, the underlying mechanism needs to be Cancers 2020, 12, 2661 11 of 13 further explored. Moreover, based on the finding by Deng et al. [11], we suggest the use of anti-HLA-G therapy to compare the results with anti-PD-L1 therapy in combination with CDK4/6 inhibitors. Finally, proliferation index of tumor (Ki-67 index) is found in correlation with mitotic index in differentiated tumors that helps identify low and high grade tumors [27]. Furthermore, we have shown here that HLA-G and PD-L1 increase during mitosis; these findings suggest that Ki-67 may also be used as a marker of immune resistance in tumor nodules. Recently, Patel et al. showed that high-grade tumors that have higher levels of Ki-67 are more responsive to immune therapies like anti-PD-L1 or anti-CTLA-4 [28]. Therefore, further studies taking these findings in account may be helpful in pre-screening of the cancer patients for immune therapy. In conclusion, our results indicate that cancer cells in a mitotic state resist against immune cell attack via higher expression of immune checkpoint molecules, suggesting the impact of the mitosis index for cancer immunotherapy.

5. Conclusions In conclusion, the cancer cells in mitosis resist to immune cells attack. This phenomena is highly dependent on HLA-G expression during cell division. These results suggest the use of proliferation index (Ki-67), which is directly related to mitotic index, as a marker of immune resistance. In addition, the use of mitotic inhibitors may result in increased immune resistance, therefore should be avoided. However, the cells in G1 phase express lower levels of HLA-G as well as PD-L1, therefore cell cycle inhibitors that block cells in G1 phase should be preferred to lower immune resistance.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/9/2661/s1, Figure S1: Immunofluorescence for HLA-G and PD-L1 protein expression, Figure S2: Explanatory figure for cell cycle analysis based on decile 2 (Figure2), Figure S3: Images captured at 15 min after co-culture of BMNCs (Plain arrow; small round cells) with OVCAR-3 cells (Dotted Arrow; with stained Nuclei in blue), Figure S4: FACS overlay peaks for Isotype and specific staining, Video S1: OVCAR-3 cell were co-cultured with BMNCs as explained in materials and methods. Author Contributions: M.U.: conceptualization, investigation, visualization and writing—original draft, W.A.: investigation, C.P.: methodology and investigation, M.P.: Supervision and interpretation, M.M.: Supervision, Conceptualization, Visualization and Writing—Original draft and review & editing. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The authors would like to thanks Amu Therwath for help in improvement of the text, and Alix Jacquier (Hôpital Saint-Louis) for her valuable assistance. Conflicts of Interest: The authors report no conflict of interest of any kind.

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