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Tulotta et al. J Cancer Metastasis Treat 2019;5:74 I http://dx.doi.org/10.20517/2394-4722.2019.022 Page 7 of 11

cancer cells pre-treated in vitro with the CXCR4 antagonist IT1t displayed reduced metastatic potential in
zebrafish. Impaired tumour burden in vivo was also observed upon genetic inhibition of tumour-derived
CXCR4 or microenvironment-dependent Cxcl12. In conclusion, we showed that the xenograft approach
in zebrafish is a valuable model to study human tumours as the CXCR4 signalling functions in human
cells upon zebrafish CXCL12 stimulation and vice versa CXCR4-expressing zebrafish cells respond to the
human cognate chemokine.

HOST-DEPENDENT CXCR4 SIGNALLING: CXCR4 CONTROLS THE TUMOUR METASTATIC

NICHE PREPARATION, BY REGULATING INTRINSIC NEUTROPHIL FUNCTION AND RESPONSE
TO CANCER CELLS
Immune cells are programmed to recognise and eliminate transformed cells. However, cancer cells have
evolved mechanisms that reprogram the immune defence and make the foe-to-friend switch an important
support for survival and progression. The combination of chemotherapy and immunotherapy is a current
[94]
[95]
strategy in the clinic . Galluzzi et al. have recently reviewed anti-cancer therapies that re-activate
the immune system, such as tumour-targeting antibodies, adoptive cell transfer and oncolytic viruses
(all classified as passive immunotherapy), dendritic cell-based immunotherapies, anti-cancer vaccines,
immune-stimulatory cytokines, immunomodulatory antibodies, inhibitors of immunosuppressive
metabolism, pattern recognition receptor agonist, and immunogenic cell death inducers (all classified as
active immunotherapy). Antibodies against CXCR4 are included in immunotherapeutic agents that skew
[95]
the balance between M2/M1 TAMs toward the pro-inflammatory and anti-tumour M1 phenotype .
We have recently shown the role of the host dependent CXCR4 signalling in supporting early metastatic
events in the zebrafish xenograft model. Previous work from our group has shown that neutrophils are
involved in the metastatic niche preparation by conditioning the ECM during their apparent random walk
in the transmigration from the CHT (caudal hematopoietic tissue, transient hematopoietic site) to the tail
[60]
tissue of zebrafish embryos . Because CXCR4 is known to regulate the retention of hematopoietic stem
[96]
progenitor cells (HSPCs) and differentiated leukocytes in the bone marrow in mammals , and is highly
[97]
expressed in zebrafish myeloid cells , we hypothesised that CXCR4 signalling plays a role in controlling
intrinsic neutrophil motility in physiological conditions. We found that neutrophils display altered motility
and their number fluctuates during embryo development, leading to the conclusion that CXCR4 regulates
neutrophil development in zebrafish. Moreover, a link between CXCR4 signalling and neutrophil response
[98]
during inflammation has been recently described . In our model, the neutrophilic response towards
cancer cells was also altered in zebrafish mutants with a non-functional Cxcr4 (Cxcr4b). We identified
a population of neutrophils that was mainly retained in the CHT and a population of neutrophils that
even if moving in the tissue, displayed the inability to infiltrate tumour cell aggregates in the tail fin of
Cxcr4b-null mutants. In the surrounding of cancer cells, cxcr4b-expressing neutrophils reduced their
speed in motility, while Cxcr4b-null neutrophils maintained similar speeds as in neutrophils that had not
been challenged by cancer cells. Therefore, we propose that Cxcr4 controls neutrophil development and
response to tumour cells, initiating early metastatic events [Figure 3A and C]. RNA sequencing performed
on sorted neutrophils from wild-type or cxcr4b-/- zebrafish larvae supported our conclusion that motility
[99]
and adhesion are altered when neutrophils lack a functional Cxcr4 signalling . In conclusion, we propose
that these alterations are responsible for the impaired tumour niche preparation and inhibition of early
micrometastasis formation in different types of cancer.

CONCLUSION
Cancer is a complex, multi-step disease and the second leading cause of death worldwide [1 in 6 deaths is
due to cancer, 9.6 million cancer-related deaths in 2018 (www.who.int, October 2019)]. Patients diagnosed