Head and neck squamous cell carcinomas (HNSCC) belong among severe and highly complex malignant diseases showing a high level of heterogeneity and consequently also a variance in therapeutic response, regardless of clinical stage. Our study implies that the progression of HNSCC may be supported by cancer-associated fibroblasts (CAFs) in the tumour microenvironment (TME) and the heterogeneity of this disease may lie in the level of cooperation between CAFs and epithelial cancer cells, as communication between CAFs and epithelial cancer cells seems to be a key factor for the sustained growth of the tumour mass. In this study, we investigated how CAFs derived from tumours of different mRNA subtypes influence the proliferation of cancer cells and their metabolic and biomechanical reprogramming. We also investigated the clinicopathological significance of the expression of these metabolism-related genes in tissue samples of HNSCC patients to identify a possible gene signature typical for HNSCC progression. We found that the right kind of cooperation between cancer cells and CAFs is needed for tumour growth and progression, and only specific mRNA subtypes can support the growth of primary cancer cells or metastases. Specifically, during coculture, cancer cell colony supporting effect and effect of CAFs on cell stiffness of cancer cells are driven by the mRNA subtype of the tumour from which the CAFs are derived. The degree of colony-forming support is reflected in cancer cell glycolysis levels and lactate shuttle-related transporters.

BIOCEV 1st Faculty of Medicine Charles University Prumyslova 595 25250 Vestec Czech Republic

Department of Chemistry and Biochemistry Mendel University in Brno Zemedelska 1 61300 Brno Czech Republic

Department of Otorhinolaryngology and Head and Neck Surgery 1st Faculty of Medicine University Hospital Motol Charles University 5 Uvalu 84 15006 Prague Czech Republic

Department of Pathological Physiology Faculty of Medicine Masaryk University Kamenice 5 62500 Brno Czech Republic

Department of Pathology and Molecular Medicine 2nd Faculty of Medicine University Hospital Motol Charles University 5 Uvalu 84 15006 Prague Czech Republic

Department of Physiology Faculty of Medicine Masaryk University Kamenice 5 62500 Brno Czech Republic

Max Planck Institute for the Science of Light Staudtstraße 2 91058 Erlangen Germany

Zobrazit více v PubMed

Stewart B.W., Wild C.P. World Cancer Report 2014. WHO Press; Geneva, Switzerland: 2014.

Peltanova B., Raudenska M., Masarik M. Effect of tumor microenvironment on pathogenesis of the head and neck squamous cell carcinoma: A systematic review. Mol. Cancer. 2019;18:63. doi: 10.1186/s12943-019-0983-5. PubMed DOI PMC

Ansems M., Span P.N. The tumor microenvironment and radiotherapy response; a central role for cancer-associated fibroblasts. Clin. Transl. Radiat. Oncol. 2020;22:90–97. doi: 10.1016/j.ctro.2020.04.001. PubMed DOI PMC

Gouirand V., Guillaumond F., Vasseur S. Influence of the Tumor Microenvironment on Cancer Cells Metabolic Reprogramming. Front. Oncol. 2018;8:117. doi: 10.3389/fonc.2018.00117. PubMed DOI PMC

Kumar D., New J., Vishwakarma V., Joshi R., Enders J., Lin F., Dasari S., Gutierrez W.R., Leef G., Ponnurangam S., et al. Cancer-Associated Fibroblasts Drive Glycolysis in a Targetable Signaling Loop Implicated in Head and Neck Squamous Cell Carcinoma Progression. Cancer Res. 2018;78:3769–3782. doi: 10.1158/0008-5472.CAN-17-1076. PubMed DOI PMC

Bonuccelli G., Tsirigos A., Whitaker-Menezes D., Pavlides S., Pestell R.G., Chiavarina B., Frank P.G., Flomenberg N., Howell A., Martinez-Outschoorn U.E., et al. Ketones and lactate “fuel” tumor growth and metastasis. Cell Cycle. 2010;9:3506–3514. doi: 10.4161/cc.9.17.12731. PubMed DOI PMC

Erdogan B., Ao M., White L.M., Means A.L., Brewer B.M., Yang L., Washington M.K., Shi C., Franco O.E., Weaver A.M., et al. Cancer-associated fibroblasts promote directional cancer cell migration by aligning fibronectin. J. Cell Biol. 2017;216:3799–3816. doi: 10.1083/jcb.201704053. PubMed DOI PMC

Wen S., Hou Y., Fu L., Xi L., Yang D., Zhao M., Qin Y., Sun K., Teng Y., Liu M. Cancer-associated fibroblast (CAF)-derived IL32 promotes breast cancer cell invasion and metastasis via integrin β3–p38 MAPK signalling. Cancer Lett. 2018;442:320–332. doi: 10.1016/j.canlet.2018.10.015. PubMed DOI

Mierke C.T. Mechanical Cues Affect Migration and Invasion of Cells From Three Different Directions. Front. Cell Dev. Biol. 2020;8:946. doi: 10.3389/fcell.2020.583226. PubMed DOI PMC

Zanotelli M.R., Rahman-Zaman A., VanderBurgh J.A., Taufalele P.V., Jain A., Erickson D., Bordeleau F., Reinhart-King C.A. Energetic costs regulated by cell mechanics and confinement are predictive of migration path during decision-making. Nat. Commun. 2019;10:4185. doi: 10.1038/s41467-019-12155-z. PubMed DOI PMC

Lawrence M.S., Sougnez C., Lichtenstein L., Cibulskis K., Lander E., Gabriel S.B., Getz G., Ally A., Balasundaram M., Birol I., et al. Comprehensive genomic characterization of head and neck squamous cell carcinomas. Nature. 2015;517:576–582. PubMed PMC

Vicar T., Raudenska M., Gumulec J., Balvan J. The Quantitative-Phase Dynamics of Apoptosis and Lytic Cell Death. Sci. Rep. 2020;10:1566. doi: 10.1038/s41598-020-58474-w. PubMed DOI PMC

Rosendahl P., Plak K., Jacobi A., Kraeter M., Toepfner N., Otto O., Herold C., Winzi M., Herbig M., Ge Y. Real-time fluorescence and deformability cytometry. Nat. Methods. 2018;15:355. doi: 10.1038/nmeth.4639. PubMed DOI

Müller P., O’Connell E., Schloögel M. Shape-Out Version 2.6.4: Analysis Software for Real-Time Deformability Cytometry [Software] Volume 2021. Max Planck Institute; Dresden, Germany: 2021. [(accessed on 28 April 2020)]. Available online: https://github.com/ZELLMECHANIK-DRESDEN/ShapeOut2.

R Core Team . R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing; Vienna, Austria: 2020.

Wickham H. Ggplot2: Elegant Graphics for Data Analysised. Springer; New York, NY, USA: 2016.

Warnes G.R., Bolker B., Bonebakker L., Gentleman R., Huber W., Liaw A., Lumley T., Maechler M., Magnusson A., Moeller S., et al. Gplots: Various R Programming Tools for Plotting Data. ScienceOpen; Berlin, Germany: 2020.

Day A. Heatmap.Plus: Heatmap with More Sensible Behavior. 2012. [(accessed on 25 March 2022)]. Available online: https://rdrr.io/cran/heatmap.plus/

Wei T., Simko V. R Package “Corrplot”: Visualization of a Correlation Matrix. 2017. [(accessed on 25 March 2022)]. Available online: https://scirp.org/reference/referencespapers.aspx?referenceid=3067218.

Kassambara A. Ggpubr: ‘ggplot2’ Based Publication Ready Plots. 2020. [(accessed on 25 March 2022)]. Available online: https://github.com/kassambara/ggpubr.

Venables W.N., Ripley B.D. Modern Applied Statistics with S. 4th ed. Springer; New York, NY, USA: 2002.

Therneau T.M., Grambsch P.M. Modeling Survival Data: Extending the Cox Modeled. Springer; New York, NY, USA: 2000.

Kassambara A., Kosinski M., Biecek P. Survminer: Drawing Survival Curves Using ‘ggplot2’. R Package Version 0.4.9. [(accessed on 25 March 2022)];2021 Volume 2021 Available online: https://rpkgs.datanovia.com/survminer/

Schliekelman M.J., Creighton C.J., Baird B.N., Chen Y., Banerjee P., Bota-Rabassedas N., Ahn Y.H., Roybal J.D., Chen F., Zhang Y., et al. Thy-1(+) Cancer-associated Fibroblasts Adversely Impact Lung Cancer Prognosis. Sci. Rep. 2017;7:6478. doi: 10.1038/s41598-017-06922-5. PubMed DOI PMC

Wirtz E.D., Hoshino D., Maldonado A.T., Tyson D.R., Weaver A.M. Response of head and neck squamous cell carcinoma cells carrying PIK3CA mutations to selected targeted therapies. JAMA Otolaryngol. Head Neck Surg. 2015;141:543–549. doi: 10.1001/jamaoto.2015.0471. PubMed DOI PMC

Chen J., Zhou J., Lu J., Xiong H., Shi X., Gong L. Significance of CD44 expression in head and neck cancer: A systemic review and meta-analysis. BMC Cancer. 2014;14:15. doi: 10.1186/1471-2407-14-15. PubMed DOI PMC

Mierke C.T. The Role of the Optical Stretcher Is Crucial in the Investigation of Cell Mechanics Regulating Cell Adhesion and Motility. Front. Cell Dev. Biol. 2019;7:184. doi: 10.3389/fcell.2019.00184. PubMed DOI PMC

Guck J., Schinkinger S., Lincoln B., Wottawah F., Ebert S., Romeyke M., Lenz D., Erickson H.M., Ananthakrishnan R., Mitchell D., et al. Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence. Biophys. J. 2005;88:3689–3698. doi: 10.1529/biophysj.104.045476. PubMed DOI PMC

Magan M., Wiechec E., Roberg K. CAFs affect the proliferation and treatment response of head and neck cancer spheroids during co-culturing in a unique in vitro model. Cancer Cell Int. 2020;20:599. doi: 10.1186/s12935-020-01718-6. PubMed DOI PMC

Biffi G., Tuveson D.A. Diversity and Biology of Cancer-Associated Fibroblasts. Physiol. Rev. 2021;101:147–176. doi: 10.1152/physrev.00048.2019. PubMed DOI PMC

Nielsen S.R., Quaranta V., Linford A., Emeagi P., Rainer C., Santos A., Ireland L., Sakai T., Sakai K., Kim Y.S., et al. Macrophage-secreted granulin supports pancreatic cancer metastasis by inducing liver fibrosis. Nat. Cell Biol. 2016;18:549–560. doi: 10.1038/ncb3340. PubMed DOI PMC

Zhang G., Zhang Y., Dong D., Wang F., Ma X., Guan F., Sun L. MCT1 regulates aggressive and metabolic phenotypes in bladder cancer. J. Cancer. 2018;9:2492–2501. doi: 10.7150/jca.25257. PubMed DOI PMC

Salt M.B., Bandyopadhyay S., McCormick F. Epithelial-to-mesenchymal transition rewires the molecular path to PI3K-dependent proliferation. Cancer Discov. 2014;4:186–199. doi: 10.1158/2159-8290.CD-13-0520. PubMed DOI

Chen Y.-Q., Lan H.-Y., Wu Y.-C., Yang W.-H., Chiou A., Yang M.-H. Epithelial-mesenchymal transition softens head and neck cancer cells to facilitate migration in 3D environments. J. Cell. Mol. Med. 2018;22:3837–3846. doi: 10.1111/jcmm.13656. PubMed DOI PMC

De Smet F., Saiz Rubio M., Hompes D., Naus E., De Baets G., Langenberg T., Hipp M.S., Houben B., Claes F., Charbonneau S., et al. Nuclear inclusion bodies of mutant and wild-type p53 in cancer: A hallmark of p53 inactivation and proteostasis remodelling by p53 aggregation. J. Pathol. 2017;242:24–38. doi: 10.1002/path.4872. PubMed DOI

Xu J., Reumers J., Couceiro J.R., De Smet F., Gallardo R., Rudyak S., Cornelis A., Rozenski J., Zwolinska A., Marine J.-C., et al. Gain of function of mutant p53 by coaggregation with multiple tumor suppressors. Nat. Chem. Biol. 2011;7:285–295. doi: 10.1038/nchembio.546. PubMed DOI

Viticchiè G., Agostini M., Lena A.M., Mancini M., Zhou H., Zolla L., Dinsdale D., Saintigny G., Melino G., Candi E. p63 supports aerobic respiration through hexokinase II. Proc. Natl. Acad. Sci. USA. 2015;112:11577–11582. doi: 10.1073/pnas.1508871112. PubMed DOI PMC

He Z., Agostini M., Liu H., Melino G., Simon H.U. P73 regulates basal and starvation-induced liver metabolism in vivo. Oncotarget. 2015;6:33178–33190. doi: 10.18632/oncotarget.5090. PubMed DOI PMC

Kim M.S., Li S.L., Bertolami C.N., Cherrick H.M., Park N.H. State of p53, Rb and DCC tumor suppressor genes in human oral cancer cell lines. Anticancer Res. 1993;13:1405–1413. PubMed

Mukhopadhyay T., Roth J.A. A codon 248 p53 mutation retains tumor suppressor function as shown by enhancement of tumor growth by antisense p53. Cancer Res. 1993;53:4362–4366. PubMed

Qiu W., Schönleben F., Li X., Su G.H. Disruption of transforming growth factor beta-Smad signaling pathway in head and neck squamous cell carcinoma as evidenced by mutations of SMAD2 and SMAD4. Cancer Lett. 2007;245:163–170. doi: 10.1016/j.canlet.2006.01.003. PubMed DOI PMC

Lin L.-H., Chang K.-W., Cheng H.-W., Liu C.-J. SMAD4 Somatic Mutations in Head and Neck Carcinoma Are Associated with Tumor Progression. Front. Oncol. 2019;9:1379. doi: 10.3389/fonc.2019.01379. PubMed DOI PMC

Wang F., Xia X., Yang C., Shen J., Mai J., Kim H.-C., Kirui D., Kang Y., Fleming J.B., Koay E.J., et al. SMAD4 Gene Mutation Renders Pancreatic Cancer Resistance to Radiotherapy through Promotion of Autophagy. Clin. Cancer Res. 2018;24:3176–3185. doi: 10.1158/1078-0432.CCR-17-3435. PubMed DOI PMC

Xu H., Tian Y., Yuan X., Wu H., Liu Q., Pestell R.G., Wu K. The role of CD44 in epithelial-mesenchymal transition and cancer development. OncoTargets Ther. 2015;8:3783–3792. PubMed PMC

Cho S.H., Park Y.S., Kim H.J., Kim C.H., Lim S.W., Huh J.W., Lee J.H., Kim H.R. CD44 enhances the epithelial-mesenchymal transition in association with colon cancer invasion. Int. J. Oncol. 2012;41:211–218. PubMed

Mah E.J., Lefebvre A.E., McGahey G.E., Yee A.F., Digman M.A. Collagen density modulates triple-negative breast cancer cell metabolism through adhesion-mediated contractility. Sci. Rep. 2018;8:17094. doi: 10.1038/s41598-018-35381-9. PubMed DOI PMC

Park J.S., Burckhardt C.J., Lazcano R., Solis L.M., Isogai T., Li L., Chen C.S., Gao B., Minna J.D., Bachoo R., et al. Mechanical regulation of glycolysis via cytoskeleton architecture. Nature. 2020;578:621–626. doi: 10.1038/s41586-020-1998-1. PubMed DOI PMC

Yubero M.L., Kosaka P.M., San Paulo A., Malumbres M., Calleja M., Tamayo J. Effects of energy metabolism on the mechanical properties of breast cancer cells. Commun. Biol. 2020;3:1–9. doi: 10.1038/s42003-020-01330-4. PubMed DOI PMC

Liberti M.V., Locasale J.W. The Warburg Effect: How Does it Benefit Cancer Cells? Trends Biochem. Sci. 2016;41:211–218. doi: 10.1016/j.tibs.2015.12.001. PubMed DOI PMC

Tan A.S., Baty J.W., Dong L.F., Bezawork-Geleta A., Endaya B., Goodwin J., Bajzikova M., Kovarova J., Peterka M., Yan B., et al. Mitochondrial genome acquisition restores respiratory function and tumorigenic potential of cancer cells without mitochondrial DNA. Cell Metab. 2015;21:81–94. doi: 10.1016/j.cmet.2014.12.003. PubMed DOI

Bajzikova M., Kovarova J., Coelho A.R., Boukalova S., Oh S., Rohlenova K., Svec D., Hubackova S., Endaya B., Judasova K., et al. Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells. Cell Metab. 2019;29:399–416. doi: 10.1016/j.cmet.2018.10.014. PubMed DOI PMC

Pérez-Escuredo J., Dadhich R.K., Dhup S., Cacace A., Van Hée V., De Saedeleer C.J., Sboarina M., Rodriguez F., Fontenille M.-J., Brisson L., et al. Lactate promotes glutamine uptake and metabolism in oxidative cancer cells. Cell Cycle. 2016;15:72–83. doi: 10.1080/15384101.2015.1120930. PubMed DOI PMC

Hui S., Ghergurovich J.M., Morscher R.J., Jang C., Teng X., Lu W., Esparza L.A., Reya T., Zhan L., Guo J.Y., et al. Glucose feeds the TCA cycle via circulating lactate. Nature. 2017;551:115–118. doi: 10.1038/nature24057. PubMed DOI PMC

Dimmer K.S., Friedrich B., Lang F., Deitmer J.W., Bröer S. The low-affinity monocarboxylate transporter MCT4 is adapted to the export of lactate in highly glycolytic cells. Biochem. J. 2000;350:219–227. doi: 10.1042/bj3500219. PubMed DOI PMC

Xu J., Lu Y., Qiu S., Chen Z.-N., Fan Z. A novel role of EMMPRIN/CD147 in transformation of quiescent fibroblasts to cancer-associated fibroblasts by breast cancer cells. Cancer Lett. 2013;335:380–386. doi: 10.1016/j.canlet.2013.02.054. PubMed DOI PMC

Öhlund D., Handly-Santana A., Biffi G., Elyada E., Almeida A.S., Ponz-Sarvise M., Corbo V., Oni T.E., Hearn S.A., Lee E.J., et al. Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer. J. Exp. Med. 2017;214:579–596. doi: 10.1084/jem.20162024. PubMed DOI PMC

Valach J., Fík Z., Strnad H., Chovanec M., Plzák J., Čada Z., Szabo P., Šáchová J., Hroudová M., Urbanová M., et al. Smooth muscle actin-expressing stromal fibroblasts in head and neck squamous cell carcinoma: Increased expression of galectin-1 and induction of poor prognosis factors. Int. J. Cancer. 2012;131:2499–2508. doi: 10.1002/ijc.27550. PubMed DOI

He J., Baum L.G. Galectin interactions with extracellular matrix and effects on cellular function. Methods Enzym. 2006;417:247–256. PubMed

Wu M.H., Hong H.C., Hong T.M., Chiang W.F., Jin Y.T., Chen Y.L. Targeting galectin-1 in carcinoma-associated fibroblasts inhibits oral squamous cell carcinoma metastasis by downregulating MCP-1/CCL2 expression. Clin. Cancer Res. 2011;17:1306–1316. doi: 10.1158/1078-0432.CCR-10-1824. PubMed DOI

Glutamine and serum starvation alters the ATP production, oxidative stress, and abundance of mitochondrial RNAs in extracellular vesicles produced by cancer cells

Long-term zinc treatment alters the mechanical properties and metabolism of prostate cancer cells