13. Quantification of the length of the strands based on a z-projected image is affected by the orientation of the strand toward
the z plane. This method of quantification gives an estimation
of the overall invasive capacity of the spheroid and z-projection
could result in an underestimation of the real length values. For
more exact quantification, measurement of invasive strands can
be performed through 3D reconstruction of the spheroid with
smaller z steps.
Acknowledgements
This study was supported by MINECO/FEDER (PGC2018099645-B-I00 to X.T.), the Generalitat de Catalunya
(SGR-2017-01602 to X.T. and CERCA Program), the European
Research Council (CoG-616480 to X.T.), the European Commission (Grant Agreement SEP-210342844 to X.T.). A.L. has
received financial support through the Junior Leader Postdoctoral
Fellowship Programme from “la Caixa” Banking Foundation (LCF/BQ/PR18/11640001). T.K. is funded by MarieCurie action (HeteroCancerInvasion # 708651) and the Japanese
Strategic Young Researcher Overseas Visits Program for Accelerating Brain Circulation. D.P is supported by Breast Cancer Now
(BCN) (2013NovPR182/Breast Cancer Now (BCN)). S.C is supported by the 2017 FPI MINECO doctoral fellowship programme
(BES-2017-079847). E.S. is supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK
(FC001144), the UK Medical Research Council (FC001144) and
the Wellcome Trust (FC001144). The authors wish to acknowledge the role of the Breast Cancer Now Tissue Bank in collecting
and making available the samples used in the generation of this
publication. IBEC is recipient of a Severo Ochoa Award of Excellence from the MINECO.
References
1. Chambers AF, Groom AC, MacDonald IC
(2002) Dissemination and growth of cancer
cells in metastatic sites. Nat Rev Cancer
2:563–572. https://doi.org/10.1038/nrc865
2. Cheung KJ, Ewald AJ (2016) A collective
route to metastasis: seeding by tumor cell clusters. Science 352:167–169. https://doi.org/
10.1126/science.aaf6546
3. Konen J, Summerbell E, Dwivedi B et al
(2017) Image-guided genomics of phenotypically heterogeneous populations reveals vascular signalling during symbiotic collective cancer
invasion. Nat Commun 8:15078
4. Cheung KJ, Padmanaban V, Silvestri V et al
(2016) Polyclonal breast cancer metastases
arise from collective dissemination of keratin
14-expressing tumor cell clusters. Proc Natl
Acad Sci U S A 113:E854–E863. https://doi.
org/10.1073/pnas.1508541113
5. Mayor R, Etienne-Manneville S (2016) The
front and rear of collective cell migration. Nat
Rev Mol Cell Biol 17:97–109
6. Cheung KJ, Gabrielson E, Werb Z, Ewald AJ
(2013) Collective invasion in breast cancer
requires a conserved basal epithelial program.
Cell 155:1639–1651
3D Invasion Assay with CAFs and Cancer Cells
255
the z plane. This method of quantification gives an estimation
of the overall invasive capacity of the spheroid and z-projection
could result in an underestimation of the real length values. For
more exact quantification, measurement of invasive strands can
be performed through 3D reconstruction of the spheroid with
smaller z steps.
Acknowledgements
This study was supported by MINECO/FEDER (PGC2018099645-B-I00 to X.T.), the Generalitat de Catalunya
(SGR-2017-01602 to X.T. and CERCA Program), the European
Research Council (CoG-616480 to X.T.), the European Commission (Grant Agreement SEP-210342844 to X.T.). A.L. has
received financial support through the Junior Leader Postdoctoral
Fellowship Programme from “la Caixa” Banking Foundation (LCF/BQ/PR18/11640001). T.K. is funded by MarieCurie action (HeteroCancerInvasion # 708651) and the Japanese
Strategic Young Researcher Overseas Visits Program for Accelerating Brain Circulation. D.P is supported by Breast Cancer Now
(BCN) (2013NovPR182/Breast Cancer Now (BCN)). S.C is supported by the 2017 FPI MINECO doctoral fellowship programme
(BES-2017-079847). E.S. is supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK
(FC001144), the UK Medical Research Council (FC001144) and
the Wellcome Trust (FC001144). The authors wish to acknowledge the role of the Breast Cancer Now Tissue Bank in collecting
and making available the samples used in the generation of this
publication. IBEC is recipient of a Severo Ochoa Award of Excellence from the MINECO.
References
1. Chambers AF, Groom AC, MacDonald IC
(2002) Dissemination and growth of cancer
cells in metastatic sites. Nat Rev Cancer
2:563–572. https://doi.org/10.1038/nrc865
2. Cheung KJ, Ewald AJ (2016) A collective
route to metastasis: seeding by tumor cell clusters. Science 352:167–169. https://doi.org/
10.1126/science.aaf6546
3. Konen J, Summerbell E, Dwivedi B et al
(2017) Image-guided genomics of phenotypically heterogeneous populations reveals vascular signalling during symbiotic collective cancer
invasion. Nat Commun 8:15078
4. Cheung KJ, Padmanaban V, Silvestri V et al
(2016) Polyclonal breast cancer metastases
arise from collective dissemination of keratin
14-expressing tumor cell clusters. Proc Natl
Acad Sci U S A 113:E854–E863. https://doi.
org/10.1073/pnas.1508541113
5. Mayor R, Etienne-Manneville S (2016) The
front and rear of collective cell migration. Nat
Rev Mol Cell Biol 17:97–109
6. Cheung KJ, Gabrielson E, Werb Z, Ewald AJ
(2013) Collective invasion in breast cancer
requires a conserved basal epithelial program.
Cell 155:1639–1651
3D Invasion Assay with CAFs and Cancer Cells
255