Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Homogeneous pancreatic cancer spheroids mimic growth pattern of circulating tumor cell clusters and macrometastases: displaying heterogeneity and crater-like structure on inner layer

  • 1093 Accesses

  • 5 Citations



Pancreatic cancer 3D in vitro models including multicellular tumor spheroid (MCTS), single cell-derived tumor spheroid (SCTS), tissue-derived tumor spheroid, and organotypic models provided powerful platforms to mimic in vivo tumor. Recent work supports that circulating tumor cell (CTC) clusters are more efficient in metastasis seeding than single CTCs. The purpose of this study is to establish 3D culture models which can mimic single CTC, monoclonal CTC clusters, and the expansion of macrometastases.


Seven pancreatic ductal adenocarcinoma cell lines were used to establish MCTS and SCTS using hanging drop and ultra-low attachment plates. Spheroid immunofluorescence staining, spheroid formation assay, immunoblotting, and literature review were performed to investigate molecular biomarkers and the morphological characteristics of pancreatic tumor spheroids.


Single cells experienced different growth patterns to form SCTS, like signet ring-like cells, blastula-like structures, and solid core spheroids. However, golf ball-like hollow spheroids could also be detected, especially when DanG and Capan-1 cells were cultivated with fibroblast-conditioned medium (p < 0.05). The size of golf ball-like hollow spheroids hardly grew after getting matured. Only DanG and Capan-1 could establish SCTS- and MCTS-derived hollow spheroids using hanging drop plates and ultra-low attachment plates. Other PDA cell lines could also establish tumor spheroid with hanging drop plates by adding methylated cellulose. Tumor spheroids derived from pancreatic cancer cell line DanG possessed asymmetrically distributed proliferation center, immune-checkpoint properties. ß-catenin, Ki-67, and F-actin were active surrounding the crater-like structure distributing on the inner layer of viable rim cover of the spheroids, which was relevant to well-differentiated tumor cells.


It is possible to establish 3D CTC cluster models from homogenous PDA cell lines using hanging drop and ultra-low attachment plates. PDA cell line displays its own intrinsic properties or heterogeneity. The mechanism of formation of the crater-like structure as well as golf ball-like structure needs further exploration.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Change history

  • 16 June 2017

    An erratum to this article has been published.


  1. Aceto N et al (2014) Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis. Cell 158(5):1110–1122

  2. Baker LA et al (2016) Modeling pancreatic cancer with organoids. Trends Cancer 2(4):176–190

  3. Calvet CY, André FM, Mir LM (2014) The culture of cancer cell lines as tumorspheres does not systematically result in cancer stem cell enrichment. PLoS One 9(2):e89644

  4. Cheung KJ, Ewald AJ (2016) A collective route to metastasis: seeding by tumor cell clusters. Science 352(6282):167–169

  5. Chu MY, Naguib FN, Iltzsch MH, el Kouni MH, Chu SH, Cha S, Calabresi P (1984) Potentiation of 5-fluoro-2′- deoxyuridine antineoplastic activity by the uridine phosphorylase inhibitors benzylacyclouridine and benzyloxybenzylacyclouridine. Cancer Res 44(5):1852–1856

  6. Deer EL, Gonzalez-Hernandez J, Coursen JD, Shea JE, Ngatia J, Scaife CL, Firpo MA, Mulvihill SJ (2010) Phenotype and genotype of pancreatic cancer cell lines. Pancreas 39:425–435

  7. Dolznig H, Rupp C, Puri C, Haslinger C, Schweifer N, Wieser E, Kerjaschki D, Garin-Chesa P (2011) Modeling colon adenocarcinomas in vitro: A 3D co-culture system induces cancer-relevant pathways upon tumor cell and stromal fibroblast interaction. Am J Pathol 179(1):487–501

  8. Dormann S, Deutsch A (2002) Modeling of self-organized avascular tumor growth with a hybrid cellular automaton. In Silico Biol 2(3):393–406

  9. Fanjul M, Hollande E (1993) Morphogenesis of “duct-like” structures in three-dimensional cultures of human cancerous pancreatic duct cells (Capan-1). Vitro Cell Dev Biol -Anim 29(7):574–584

  10. Farnie G et al (2007) Novel cell culture technique for primary ductal carcinoma in situ: role of Notch and epidermal growth factor receptor signaling pathways. J Natl Cancer Ins 99(8):616–627

  11. Fredebohm J, Boettcher M, Eisen C, Gaida MM, Heller A, Keleg S, Tost J et al (2012) Establishment and characterization of a highly tumourigenic and cancer stem cell enriched pancreatic cancer cell line as a well defined model system. PLoS One 7(11):e48503

  12. Haselmann V, Kurz A, Bertsch U, Hübner S, Olempska-Müller M, Fritsch J, Häsler R et al (2014) Nuclear death receptor TRAIL-R2 inhibits maturation of let-7 and promotes proliferation of pancreatic and other tumor cells. Gastroenterology 146(1):278–290

  13. Hirai Y, Nelson CM, Yamazaki K, Takebe K, Przybylo J, Madden B, Radisky DC (2007) Non-classical export of epimorphin and its adhesion to αv-integrin in regulation of epithelial morphogenesis. J Cell Sci 120(12):2032–2043

  14. Hirose M et al (1996) A new function of Ito cells in liver morphogenesis: evidence using a novel morphogenic protein, epimorphin, in vitro. Biochem Biophys Res Commun 225:155–160

  15. Humphrey ES, Su S-P, Nagrial AM, Hochgrafe F, Pajic M, Lehrbach GM, Parton RG, Yap AS, Horvath LG, Chang DK, Biankin AV, Wu J, Daly RJ (2016) Resolution of novel pancreatic ductal adenocarcinoma subtypes by global phosphotyrosine profiling. Mol Cell Proteom 15:2671–2685

  16. Kalthoff H, Roeder C, Humburg I, Thiele H-G, Greten H, Schmiegel W (1991) Modulation of platelet-derived growth factor A- and B-chain/c-sis mRNA by tumor necrosis factor and other agents in adenocarcinoma cells. Oncogene 6:1015–1021

  17. Kang H-G, Jenabi JM, Zhang J, Keshelava N, Shimada H, May WA, Ng T, Reynolds CP, Triche TJ, Sorensen PHB (2007) E-cadherin cell-cell adhesion in ewing tumor cells mediates suppression of anoikis through activation of the ErbB4 tyrosine kinase. Cancer Res 67(7):3094–3105

  18. Kato N, Narutomi K, Fukase M, Motoyama T (2012) Hollow spheroids in ascites of ovarian clear cell carcinoma: how are they formed and how do they behave? Cytopathology 23(2):120–125

  19. Koshida S, Hirai Y (1997) Identification of cellular recognition sequence of epimorphin and critical role of cell/epimorphin interaction in lung branching morphogenesis. Biochem Biophys Res Commun 234:522–525

  20. Lee JM, M-F P, Lee N, Parsanian LC, Lin YG, Gayther SA, Lawrenson K (2013) A three-dimensional microenvironment alters protein expression and chemosensitivity of epithelial ovarian cancer cells in vitro. Lab Invest 93:528–542

  21. Lehnert L et al (1999) Hollow-spheroids: a new model for analyses of differentiation of pancreatic duct epithelial cells. Ann NY Acad Sci 880(1):83–93

  22. Lehnert L et al (2001) Autocrine stimulation of human pancreatic duct—like development by soluble isoforms of epimorphin in vitro. J Cell Biol 152(5):911–922

  23. Li Y, Arao Y, Hall JM, Burkett S, Liu L, Gerrish K, Cavailles V, Korach KS (2014) Research resource: STR DNA profile and gene expression comparisons of human BG-1 cells and a BG-1/MCF-7 clonal variant. Mol Endocrinol 28:2072–2081

  24. Little JL, Serzhanova V, Izumchenko E, Egleston BL, Parise E, Klein-Szanto AJ, Loudon G et al (2014) A requirement for Nedd9 in luminal progenitor cells prior to mammary tumorigenesis in MMTV-HER2/ErbB2 mice. Oncogene 33(4):411–420

  25. Liu JC, Deng T, Lehal RS, Kim J, Zacksenhaus E (2007) Identification of tumorspheroid-and tumor-initiating cells in HER2/Neu-induced mammary tumors. Can Res 67(18):8671–8681

  26. Maddipati R, Stanger BZ (2015) Pancreatic cancer metastases harbor evidence of polyclonality. Cancer Discov 5(10):1086–1097

  27. Mayer B, Klement G, Kaneko M, Man S, Jothy S, Rak J, Kerbel RS (2001) Multicellular gastric cancer spheroids recapitulate growth pattern and differentiation phenotype of human gastric carcinomas. Gastroenterology 121(4):839–852

  28. Mori M, Miyazaki K (2000) Factors affecting morphogenesis of rabbit gallbladder epithelial cells cultured in collagen gels. Cell Tissue Res 300:331–344

  29. Neureiter D, Zopf S, Dimmler A, Stintzing S, Hahn EG, Kirchner T, Herold C, Ocker M (2005) Different capabilities of morphological pattern formation and its association with the expression of differentiation markers in a xenograft model of human pancreatic cancer cell lines. Pancreatology 5(4–5):387–397

  30. O’Brien LE et al (2002) Building epithelial architecture: insights from three-dimensional culture models. Nat Rev Mol Cell Biol 3:531–537

  31. Radisky DC, Hirai Y, Bissell MJ (2003) Delivering the message: epimorphin and mammary epithelial morphogenesis. Trends Cell Biol 13(8):426–434

  32. Rejniak KA (2007) An immersed boundary framework for modelling the growth of individual cells: an application to the early tumour development. J Theor Biol 247(1):186–204

  33. Rejniak KA (2012) Homeostatic imbalance in epithelial ducts and its role in carcinogenesis. Scientifica 2012:132978

  34. Rejniak KA, Dillon RH (2007) A single cell-based model of the ductal tumour microarchitecture. Comput Math Methods Med 8(1):51–69

  35. Rejniak KA, Kliman HJ, Fauci LJ (2004) A computational model of the mechanics of growth of the villous trophoblast bilayer. Bull Math Biol 66(2):199–232

  36. Schmiegel W, Roeder C, Schmielau J, Rodeck U, Kalthoff H (1993) Tumor necrosis factor alpha induces the expression of transforming growth factor alpha and the epidermal growth factor receptor in human pancreatic cancer cells. Proc Natl Acad Sci USA 90:863–867

  37. Siegel RL, Miller KD, Jemal A (2016) Cancer statistics, 2016. CA Cancer J Clin 66(1):7–30

  38. Sirivatanauksorn V, Sirivatanauksorn Y, Gorman PA, Davidson JM, Sheer D, Moore PS, Scarpa A, Edwards PAW, Lemoine NR (2001) Non-random chromosomal rearrangements in pancreatic cancer cell lines identified by spectral karyotyping. Int J Cancer 91:350–358

  39. Smart CE, Morrison BJ, Saunus JM, Vargas AC, Keith P, Reid L, Wockner L et al (2013) In vitro analysis of breast cancer cell line tumourspheres and primary human breast epithelia mammospheroids demonstrates inter-and intraspheroid heterogeneity. PLoS One 8(6):e64388

  40. Tamura K, Yokoyama S, Ieda J, Takifuji K, Hotta T, Matsuda K, Oku Y et al (2011) Hollow spheroids beyond the invasive margin indicate the malignant potential of colorectal cancer. BMJ Open 1(1):e000179

  41. Uhlén M, Fagerberg L, Hallström BM, Lindskog C, Oksvold P, Mardinoglu A, Sivertsson Å et al (2015) Tissue-based map of the human proteome. Science 347(6220):1260419

  42. Vinci M et al (2012) Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biol 10(1):1

  43. Wang R, Lv Q, Meng W, Tan Q, Zhang S, Mo X, Yang X (2014) Comparison of mammospheroid formation from breast cancer cell lines and primary breast tumors. J Thor Dis 6(6):829–837

  44. Ware MJ et al (2016) Generation of homogenous three-dimensional pancreatic cancer cell spheroids using an improved hanging drop technique. Tissue Eng Part C: Methods 22(4):312–321

  45. Weiswald L-B, Bellet D, Dangles-Marie V (2015) Spherical cancer models in tumor biology. Neoplasia 17(1):1–15

  46. Winterhoff BJN, Arlt A, Duttmann A, Ungefroren H, Schäfer H, Kalthoff H, Kruse M-L (2012) Characterisation of FAP-1 expression and CD95 mediated apoptosis in the A818-6 pancreatic adenocarcinoma differentiation system. Differentiation 83(3):148–157

  47. Wu X, Zahari MS, Renuse S, Nirujogi RS, Kim M-S, Manda SS, Stearns V, Gabrielson E, Sukumar S, Pandey A (2015) Phosphoproteomic analysis identifies focal adhesion kinase 2 (FAK2) as a potential therapeutic target for tamoxifen resistance in breast cancer. Mol Cell Proteom 14:2887–2900

  48. Yeon S-E et al (2013) Application of concave microwells to pancreatic tumor spheroids enabling anticancer drug evaluation in a clinically relevant drug resistance model. PloS one 8(9):e73345

Download references


The study was supported by China Scholarship Council (201306230127). The authors would like to thank for the platform support of University Hospital of LMU Munich for the preliminary experiments.

Author information

Correspondence to Hao Feng or Wolfgang E. Thasler.

Ethics declarations

Conflict of interest

There is no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

An erratum to this article is available at https://doi.org/10.1007/s00432-017-2456-9.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Feng, H., Ou, B., Zhao, J. et al. Homogeneous pancreatic cancer spheroids mimic growth pattern of circulating tumor cell clusters and macrometastases: displaying heterogeneity and crater-like structure on inner layer. J Cancer Res Clin Oncol 143, 1771–1786 (2017). https://doi.org/10.1007/s00432-017-2434-2

Download citation


  • Pancreatic cancer
  • Tumor spheroid
  • Heterogeneity
  • Differentiation
  • Circulating tumor cell