Evolution of placental invasion and cancer metastasis are causally linked

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Among mammals, placental invasion is correlated with vulnerability to malignancy. Animals with more invasive placentation (for example, humans) are more vulnerable to malignancy. To explain this correlation, we propose the hypothesis of ‘Evolved Levels of Invasibility’ proposing that the evolution of invasibility of stromal tissue affects both placental and cancer invasion. We provide evidence for this using an in vitro model. We find that bovine endometrial and skin fibroblasts are more resistant to invasion than are their human counterparts. Gene expression profiling identified genes with high expression in human but not in bovine fibroblasts. Knocking down a subset of them in human fibroblasts leads to stronger resistance to cancer cell invasion. Identifying the evolutionary determinants of stromal invasibility can provide important insights to develop rational antimetastatic therapeutics.

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Fig. 1: An experimental platform to test ELI.
Fig. 2: Bovine stroma resists trophoblast and melanoma invasion.
Fig. 3: Transcriptomic analysis of bovine and human ESFs reveals differential response to trophoblasts.
Fig. 4: Induced resistance to invasion in human stroma by evolutionarily inspired gene silencing.

Data availability

Data for the human–cow transcriptome comparison with and without co-culture with trophoblast cells are available under GSE136299 in the Gene Expression Omnibus (GEO) database of NCBI, https://www.ncbi.nlm.nih.gov/geo/. The comparative fibroblast gene expression data are available under PRJNA564062 under SUB6229748 and SUB6264591 on the Sequence Read Archive (SRA) of NCBI, https://www.ncbi.nlm.nih.gov/sra.

Code availability

The computational methods are described and cited in Methods. No new code was used in this study.


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This work was funded by National Cancer Institute Center grant no. U54-CA209992.

Author information

G.P.W. and A.L. conceptualized this project. K., J.D.M. and C.L. were responsible for data curation. K., C.L. and G.P.W. conducted the formal analysis. A.L. and G.P.W. acquired the funding. K., E.E., J.D.M. and A.H. undertook the investigation. K. and H.N. were responsible for the methodology. A.L. and G.P.W. were responsible for project admininstration. J.-D.H., C.P., T.H., T.O., T.S., M.P. and D.F.A. obtained resources. C.L. worked on software. G.P.W. and A.L. supervised the project. K., E.M.E. and G.P.W. validated the results. K. and C.L. worked on visualization. K., G.P.W. and A.L. wrote the first draft and all other authors were involved in reviewing and editing.

Correspondence to Kshitiz or Andre Levchenko or Günter P. Wagner.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Nanotextured stromal invasion assay quantitatively and sensitively measures collective intrusion of cells into stroma.

(A) Time stamped images at 0 and 18 hours showing extent of invasion by DiI-labelled 1205Lu cells (red) into BJ5ta stromal fibroblast monolayer (unlabelled) on flat and nanotextured substrata; Quantification shown in Fig. 1d. (B-C) Sensitive measurement of differences in invasion by nanotextured stromal invasion platform; Time stamped images at 0, 6, 12 and 18 hours showing extent of invasion by DiI-labelled non-malignant WM35 (B), and malignant 1205Lu (C) cells into BJ5ta monolayer shown in both flat, and nanotextured cell patterned assay; Quantification shown in Fig. 1e.

Extended Data Fig. 2 Human and bovine trophoblasts exhibit different invasive potential into endometrial stroma.

Time stamped images showing extent of invasion of human choriocarcinoma-derived trophoblasts, J3 (red) (A), and bovine trophoblasts, F3 (red) (B) into their respective endometrial stromal fibroblast monolayers at 0, and 48 hours.

Extended Data Fig. 3 Human and bovine ESFs respond differently to co-culture with trophoblasts.

(A) Experimental plan to isolate endometrial cells after co-culture with respective trophoblast; (B) Volcano plot showing fold change in genes between bovine versus human endometrial stromal fibroblasts, along with their significance depicted in P value. (C) P-value distribution of t-tests comparing human ESFs with and without co-culture with HTR8 trophoblast cells. (D) P-value distribution of t-tests comparing bovine ESFs with and without co-culture with F3 trophoblast cells. In both C and D, note that thin right hand tail of the distribution, which indicates that a large number of genes are differentially expressed in response to the presence of the corresponding trophoblast cells. (E-F) Scatter plots showing relative TPM values of genes between (E) hESF and hESF co-cultured with HTR8, and (F) bESF and bESF co-cultured with F3; Red dots refer to individual gene transcripts abundance significantly different in between the compared conditions.

Extended Data Fig. 4 Human and bovine endometrial stromal fibroblasts do not appear to differentially respond to their respective trophoblasts by regulating cell-cell adhesion.

Gene ontology analysis of individual genes belonging to GO_Adherens (A), GO_Endothelial Barrier (B), GO_Fibroblast Migration (C), and Kegg ontology for Wnt Signalling (D) for hESF and bESF shown with their relative TPM values; Also are shown the coefficient of determinant, R2 for the linear regression between TPM values of hESF and bESF in a given gene-sets, along with the standard deviation of the residuals, Sy.x. (E) Ingenuity Pathway Analysis of selected signalling pathways differentially activated in hESF and bESF; Shown are –log(p-values) of genes in the respective pathways differentially activated, while the colour depicts the z-score.

Extended Data Fig. 5 siRNA-induced gene knockdown reduces transcript levels in hESFs and BJ5ta cells.

qRT–PCR results showing the percentage knockdown of transcript levels in hESF (A) and BJ5ta (B), calculated using ΔΔCt method, compared using GAPDH as housekeeping gene with scrambled siRNA as control. For both A, and B, n = 4 biological replicates.

Supplementary information

Reporting Summary

Supplementary Video 1

bESF versus F3-mCherry: imaging of mCherry labelled F3 bovine trophoblast cells invading a monolayer of unlabelled bovine endometrial stromal cells. The total imaging duration is 44 h.

Supplementary Video 2

bESF versus F3-Phase: phase-contrast-based imaging of mCherry labelled F3 bovine trophoblast cells invading a monolayer of unlabled bovine endometrial stromal cells. The total imaging duration is 44 h.

Supplementary Video 3

hESF versus J3-mCherry: imaging of mCherry labelled J3 human choriocarcinoma cells invading a monolayer of human endometrial stromal cells. The total imaging duration is 44 h.

Supplementary Video 4

hESF versus J3-Phase: phase-contrast-based imaging of mCherry labelled J3 human choriocarcinoma cells invading a monolayer of human endometrial stromal cells. The total imaging duration is 44 h.

Supplementary Video 5

hESF versus BeWo: imaging of cytotracker green labelled HTR8 invading into a monolayer of unlabelled hESFs. The total imaging duration is 24 h.

Supplementary Video 6

hESF versus HTR8: imaging of cytotracker green labelled BeWo invading into a monolayer of unlabelled hESFs. The total imaging duration is 24 h.

Supplementary Video 7

hESF versus F3: imaging of cytotracker green labelled F3 invading into a monolayer of unlabelled hESFs. The total imaging duration is 24 h.

Supplementary Video 8

bESF versus BeWo: imaging of cytotracker green labelled HTR8 invading into a monolayer of unlabelled bESFs. The total imaging duration is 24 h.

Supplementary Video 9

bESF versus HTR8: imaging of cytotracker green labelled BeWo invading into a monolayer of unlabelled bESFs. The total imaging duration is 24 h.

Supplementary Video 10

bESF versus F3: imaging of cytotracker green labelled F3 invading into a monolayer of unlabelled bESFs. The total imaging duration is 24 h.

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Kshitiz, Afzal, J., Maziarz, J.D. et al. Evolution of placental invasion and cancer metastasis are causally linked. Nat Ecol Evol 3, 1743–1753 (2019) doi:10.1038/s41559-019-1046-4

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