Cross, Neil

Hudson, Katie

Jordan-Mahy, Nicola

Leyland, Rebecca

Sheffield Hallam University Research Archive

Investigating the tumour-intrinsic role of programmed death-ligand 1 in 2D and 3D cell culture models of human breast cancerHUDSON, Katie, CROSS, Neil <http://orcid.org/0000-0003-2055-5815>, JORDAN-MAHY, Nicola and LEYLAND, RebeccaAvailable from Sheffield Hallam University Research Archive (SHURA) at:http://shura.shu.ac.uk/29566/This document is the author deposited version.  You are advised to consult the publisher's version if you wish to cite from it.Published versionHUDSON, Katie, CROSS, Neil, JORDAN-MAHY, Nicola and LEYLAND, Rebecca (2021). Investigating the tumour-intrinsic role of programmed death-ligand 1 in 2D and 3D cell culture models of human breast cancer. In: British Society for Immunology Congress 2021, Edinburgh, UK, 28 Nov - 01 Dec 2021. (Unpublished) Copyright and re-use policySee http://shura.shu.ac.uk/information.htmlSheffield Hallam University Research Archivehttp://shura.shu.ac.uk00.0031250.006250.01250.0250.050.10.20.4 1 5 100501001502002D-cultured cellsAtezolizumab Concentration (nM)Median Fluorescence Intensity***********************00.0250.050.10.2 0.4 1 5 102002040603D spheroidsMedian Fluorescence IntensityAtezolizumab Concentration (nM)Investigating the Tumour-Intrinsic Role of Programmed Death-Ligand 1 in 2D and 3D Cell Culture Models of Human Breast CancerKatie Hudson1, Neil Cross1, Nicola Jordan-Mahy1, Rebecca Leyland11Biomolecular Sciences Research Centre, City Campus, Sheffield Hallam University, Howard Street, Sheffield, S1 1WB*corresponding author (R.Leyland@shu.ac.uk)IntroductionResearch AimConclusionsReferencesResults• Tumorigenic expression of programmed death-ligand 1 (PD-L1) is found in many cancersand is well known to promote immune evasion and tumour progression through binding toits receptor PD-1 on T cells and other immune cells (Figure 1) (Jiang et al., 2019).• Monoclonal antibodies targeting PD-L1/PD-1 promote durable anti-tumour immuneresponses in select patients with advanced cancers, but most patients are unresponsive,hyperprogressive or develop resistance (Yang and Hu, 2019).• The tumour-intrinsic role of PD-L1 and how immunotherapy treatment may influence itsrole remains poorly understood in all cancers.• So far, most research investigating tumorigenic PD-L1 and its response to treatment isbased on 2D cell culture models or immunocompromised mouse models that fail torecapitulate in vivo human tumours (Dong et al., 2018; Hudson et al., 2020).• Better mimicking of the 3D architecture of solid tumours by utilising 3D cell culture modelscould provide an environment more representative of in vivo human tumours for thepreclinical investigation of tumour-intrinsic PD-L1 signalling, response to immunotherapytreatment and cancer cell-immune cell interactions.To determine the phenotypic effects of blocking PD-L1 with an anti-PD-L1 immunotherapydrug compared to knocking down PD-L1 in human breast cancer cells cultured in 2D and 3Dcell culture models.A BJiang, X., Wang, J., Deng, X., Xiong, F., Ge, J., Xiang, B., Wu, X., MA, J., Zhou, M., Li, X., Li, Y., Li, G., Xiong, W., and Zeng, Z. (2019). Role of the tumor microenvironment in PD-L1/PD-1-mediated tumor immune escape. Molecular Cancer, 18 (1), 10. doi:10.1186/s12943-018-0928-4Yang, J., and Hu, L. (2019). Immunomodulators targeting the PD‐1/PD‐L1 protein‐protein interaction: From antibodies to small molecules. Medicinal Research Reviews, 39 (1): 265-301. doi:10.1002/med.21530Dong, P., Xiong, Y., Yue, J., Hanley, S. J. B., and Watari, H. (2018). Tumor-intrinsic PD-L1 signalling in cancer initiation, development and treatment: Beyond immune evasion. Frontiers in Oncology, 8: 386. doi:10.3389/fonc.2018.00386Hudson, K., Cross, N., Jordan-Mahy, N., and Leyland, R., (2020). The extrinsic and intrinsic roles of PD-L1 and its receptor PD-1: Implications for immunotherapy treatment. Frontiers in Immunology, 11: 568931. doi:10.3389/fimmu.2020.568931Figure 1. The new and emerging role of PD-L1 in some cancer cells. The role of PD-L1 to inhibit the immune system bybinding to its receptor PD-1 expressed by T cells and other immune cells in the tumour microenvironment is well established,but recently PD-L1 has been shown to send pro-survivals in some cancer cells which may promote cancer initiation,epithelial to mesenchymal (EMT) transition, invasion and metastasis, drug resistance and metabolic activity. It is largelyunknown whether anti-PD-L1 therapies can influence this new and emerging tumour-intrinsic role of PD-L1.Methodology1.2.3.4.MDA-MB-231, human breast cancer, wild type (WT) cells, scrambled control (SC) cells or PD-L1 knockdown (PD-L1 KD) cells2D in vitro monolayer 3D in vitro spheroidsHanging drop Alginate hydrogel beadsFluorescence MicroscopyFlow Cytometry 500 µmRNA extraction cDNA synthesis TaqMan qRT-PCRRT-qPCR100μmFSC-HSSC-HCell populationSingle cellsLive cellsFSC-AFSC-HSingle cellsPropidium IodideCell FrequencyPD-L1Cell FrequencySpheroids Spheroid ColoniesDownstream Analysis±Atezolizumab treatment ± Atezolizumab treatment CellTiter-Glo® 2D/3DCell surface staining orKi67Cell FrequencyIntracellular staining Annexin VPropidium IodideCompensation using single colour controlsAnnexin VPropidium IodideAnnexin V only controlPI only controlAnnexin VPropidium IodideLiveEarly ApoptoticLate ApoptoticNecroticGFPFSC-HGFP -cellsGFP + cellsWT/Atez treated cellsSC/PD-L1 KD cellsIsotype PD-L1Isotype Ki67Figure 2. A schematic diagram of the workflow applied to MDA-MB-231 human breast cancer cells to generate 2Dand 3D cell culture models for downstream analysis in order to determine the phenotypic effects of PD-L1 blockadecompared to PD-L1 knockdown. MDA-MB-231 WT, SC and PD-L1 KD cells were cultured in 2D and 3D cell culture. Thehanging drop method and alginate hydrogel beads were utilised to form 3D spheroids. Some cells in 2D and 3D culture weretreated with anti-PD-L1 immunotherapy drug, Atezolizumab. 2D-cultured cells and 3D hanging drop spheroids wereharvested at day 6 and 3D alginate spheroid colonies were harvested at 10 for downstream analysis. Cell viability wasassessed using Hoechst/PI staining via fluorescent microscopy, CellTiter-Glo® via plate reader and Annexin V/PI stainingvia flow cytometry. PD-L1 mRNA and protein expression was measured via RT-qPCR and flow cytometry. Cell proliferationmarker Ki67 was measured via intracellular flow cytometry staining. Appropriate controls and gating strategies were carriedout for each experiment.Isotype PD-L1 PD-L10 nM10 nM99.1%2.17%Cell Frequency 95.17%94%27.5%00.0250.050.10.20.4 1 5 10204001020303D alginate spheroid coloniesMedian Fluorescence IntensityAtezolizumab Concentration (nM)4.13%Isotype PD-L1 PD-L10 nM10 nMCell FrequencyIsotype PD-L1 PD-L10 nM10 nMCell FrequencyC2D 3D spheroids 3D spheroid colonies 0.00.51.01.52.0PD-L1 (CD274) mRNA levelsCD274 mRNA expressionrelative toHPRT1:TBP UntreatedAtezolizumabDFigure 3. Atezolizumab blocks cell surface PD-L1in a dose-dependent manner and does not effectthe level of PD-L1 mRNA in 2D and 3D cultures.Flow cytometric analysis was used to determine thecell surface expression of PD-L1 on MDA-MB-231breast cancer cells cultured in 2D and 3D followingtreatment with Atezolizumab at varyingconcentrations (0-40 nM). Representative flowcytometry plots are displayed for each culturesystem at concentrations of 0 nM and 10 nMdemonstrating how Atezolizumab can completelyblock PD-L1 in 2D-cultured cells (A) and 3Dspheroids (B), but only partially block PD-L1 in 3Dspheroid colonies. RT-qPCR was used to measurePD-L1 mRNA expression by 2D- and 3D-culturedcells following 10 nM Atezolizumab treatment.Atezolizumab treatment did not effect PD-L1 mRNAexpression (D). Data is presented as mean ± SD,n=3 independent experiments each with 3 technicalrepeats, Kruskal-Wallis followed by Conover Inmanmultiple comparison test, *P<0.05, **0.01,****P<0.0001.0.00.20.40.60.81.0CD274 expression in WT vs PD-L1 KD cellsRelativeCD274mRNA expressionnormalised toHPRT1:TBPWTScrambled ControlPD-L1 KD✱✱✱✱✱0100200300PD-L1 expression in WT vs PD-L1 KD cellsMedian Fluorescent IntensityWTScrambled ControlPD-L1 KD✱✱✱✱✱AIsotype WT Scrambled control PD-L1 KD cells GFPFSC-HGFP+ cellsGFP+/PD-L1 KD cells (FITC)GFP+/PD-L1 KD cells (Brightfield)BCFigure 4. MDA-MB-231 PD-L1 KD cells display ~70%reduction in PD-L1 expression at mRNA and proteinlevels. MDA-MB-231 cells were stably transfected with aplasmid DNA encoding GFP upstream of our PD-L1-specific knockdown strategy. Clonal assays wereperformed to isolate a colony with homogenousexpression of GFP and PD-L1 KD (A). Scale bar 100 µM.WT, SC and PD-L1 KD cells were harvested andassessed for their expression of PD-L1 at mRNA (B) andprotein (C) levels. PD-L1 KD cells express significantlylower levels of PD-L1 compared to WT and SC cells atboth mRNA and protein levels. A representative flowcytometry plot is displayed to demonstrate the levels ofPD-L1 protein expressed by WT, SC and PD-L1 KD cellscompared to the isotype control. Data is presented asmean ± SD, n=3 independent experiments each with 3technical repeats, Kruskal-Wallis followed by ConoverInman multiple comparison test, *P<0.05, ***0.001.• Atezolizumab blocks cell surface PD-L1 but does not affect PD-L1 mRNA whereas PD-L1 knockdown reduces PD-L1expression by ~70% at mRNA and protein levels• PD-L1 knockdown cells show a higher proportion of cell death and reduced proliferative capacity in 2D and 3Dmodels and are unable to form 3D spheroid colonies compared to WT and Atezolizumab-treated breast cancer cells• By using 3D models that more closely mimic the characteristics of an in vivo human tumour, we show that targetingPD-L1 at the molecular level was able to disrupt the tumorigenic functions of PD-L1 more so than AtezolizumabWTAtezolizumabScrambled ControlPD-L1 KD0204060801003D spheroid colony viability at day 10Percentage of Cells (%)NecroticLate ApoptoticEarly ApoptoticLiveWTAtezolizumabScrambled ControlPD-L1 KD01234ATP levels in 3D spheroid colonies at day 10Luminescence (RLU x 106)✱✱✱✱WTAtezolizumabScrambled ControlPD-L1 KD0204060801001203D spheroid viability at day 6Percentage of Cells (%)NecroticLate ApoptoticEarly ApoptoticLiveWTAtezolizumabScrambled ControlPD-L1 KD01234ATP levels in 3D spheroids at day 6Luminescence (RLU x 106)✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱WTAtezolizumabScrambled ControlPD-L1 KD0204060801002D-cultured cell viability at day 6Percentage of Cells (%)NecroticLate ApoptoticEarly ApoptoticLiveWTAtezolizumabScrambled ControlPD-L1 KD0246810ATP levels in 2D-cultured cells at day 6Luminescence (RLU x 106)✱✱✱✱✱✱A DBC FFigure 5. PD-L1 KD cells display a higher proportion of celldeath than WT, Atezolizumab-treated and SC cells in 2Dand 3D cultures. Flow cytometric analysis of Annexin V/PIstained WT, Atezolizumab-treated, SC and PD-L1 KD cellsreveals that PD-L1 KD cells display an higher cell deathphenotype compared to WT, Atezolizumab-treated and SC cellscultured in 2D (A), 3D spheroids (B) and 3D spheroid colonies(C). Atezolizumab induces slight changes in cell viabilitycompared to WT cells in 2D and 3D cultures. Representativeflow cytometry plots are displayed for 2D and 3D cultures whichillustrates the differences in cell viability between WT,Atezolizumab-treated, SC and PD-L1 KD cells. CellTiter-Gloexperiments were performed to validate the effects ofAtezolizumab and PD-L1 KD on cancer cell death in 2D and 3Dcultures compared to WT and/or SC cells via measuring ATPlevels. In 2D cell culture, PD-L1 KD cells display a significantdecrease in ATP levels compared to WT and Atezolizumab-treated cells but not SC cells, although still reduced (D). In 3Dspheroids, Atezolizumab-treated cells show a significantreduction in ATP levels compared to WT cells, which is evenfurther reduced in PD-L1 KD cells (E). The level of ATPproduction by cells in 3D spheroids is significantly lower in PD-L1 KD cells compared to SC cells. In 3D spheroid colonies, PD-L1 KD cells display a significant reduction in ATP productioncompared to WT, Atezolizumab-treated and SC cells which alldisplay similar levels. Data is presented as mean ± SD, n=3independent experiments each with 3 technical repeats,Kruskal-Wallis followed by Conover Inman multiple comparisontest, *P<0.05, **P<0.01, ***0.001, ****P<0.0001.PD-L1 KD cellsAtezolizumabSingle cellsCell clusterSpheroid colonies WT Scrambled controlSpheroid colonies Spheroid colonies Figure 6. PD-L1 KD cells exhibit reduced Ki67 expression in 2D and 3D models anddemonstrate an inability to form 3D spheroid colonies in the alginate bead model compared toWT, Atezolizumab-treated and SC cells. Ki67 staining reveals a lower proliferative capacity by breastcancer cells with PD-L1 knockdown compared to WT, Atezolizumab-treated and SC cells in 2D (A), 3Dspheroids (B) and 3D spheroid colonies (C). Day 10 alginate beads were stained withHoechst33342/PI and assessed for 3D spheroid colonies formation using fluorescent microscopy. PD-L1 KD cells remain single cells within the alginate compared to WT, Atezolizumab-treated and SC cellsthat form 3D spheroid colonies (D). Data is presented as mean ± SD, n=3 independent experimentseach with 3 technical repeats, normalised to WT cells either % or MFI. Scale bar represents 200 µM.0.00.51.01.5Ki67 expression by 2D-cultured cells Median Fluorescent IntensityNormalised to WT ControlIsotype WT PD-L1 KD cells AtezolizumabScrambled controlA0.00.51.01.5Ki67 expression by 3D spheroids % of Ki67 + CellsNormalised to WT ControlWTAtezolizumabScrambled ControlPD-L1 KD0.00.51.01.5Ki67 expression by 3D spheroid colonies % of Ki67 + CellsNormalised to WT ControlBDCE

Investigating the tumour-intrinsic role of programmed death-ligand 1 in 2D and 3D cell culture models of human breast cancer

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Investigating the tumour-intrinsic role of programmed death-ligand 1 in 2D and 3D cell culture models of human breast cancer

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