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Page 2 of 7 Mascarenhas et al. J Cancer Metastasis Treat 2020;6:23 I http://dx.doi.org/10.20517/2394-4722.2020.52

Keywords: Nephrilin, melanoma, metastases, deep lung, lobes, anoxia, oxidative stress, immune modulation

INTRODUCTION
The peptide nephrilin is an inhibitor of Rictor complex derived by fusing a 19 amino acid segment of
Protor with the metal-binding domain of human insulin-like growth factor binding protein-3 (IGFBP3)-a
[1-2]
21 amino acid pro-homeostatic sequence that targets and preferentially enters stressed cells .

Severe burn trauma is associated with secondary systemic effects including hyper inflammation,
hypercatabolism, sepsis, organ failure, loss of glycemic control, delayed wound healing, and cognitive
[3-8]
deficits . We have demonstrated the efficacy of nephrilin peptide in combating many of these pleiotropic
effects in burn trauma as well as in several rodent models of metabolic and xenobiotic stress [9-14] . When
nephrilin peptide is injected subcutaneously into rodents, its Protor sequence is believed to reduce
oxidative stress in target tissues by modulation of Rac1 phosphorylation and, more speculatively, its IGFBP3
sequence may mediate homeostatic immunomodulatory functions in addition to its described transporter
[12]
functions . Nephrilin treatment following burn injury reverses epigenetic and signaling changes in kidney
tissue that lead to the activation of Rac1, and lowers elevations in markers of systemic oxidative stress
such as urinary 8-isoprostane and plasma OHDG [10-12] . Besides, an analysis of gene expression in the CNS
after burn injury showed that nephrilin beneficially modulated the expression of genes associated with
[13]
astrocytosis, oxidative stress, and immunosuppression . Trauma is characterized by the co-existence of
pro-inflammatory and immunosuppression phenomena, strikingly reminiscent of what has long been
described for metastatic niche formation [15,16] . The role of oxidative stress and pro-fibrotic cytokines has
been amply documented as instrumental in the sculpting of the latter environment(s). We asked whether
nephrilin might modulate the latter environment, as we have shown that it has beneficial effects on the
former.

Previous studies have shown that the lobes of the rodent right lung differ in their access to the arterial
and lymphatic circulation, susceptibility to anoxia, and local concentrations of pro-inflammatory factors
such as lung elastase . We were interested in examining the biodistribution and effectiveness of nephrilin
[17]
peptide in the lobes of the rodent lung, to gain a better understanding of how nephrilin accesses deep
lung environments. This variable is relevant in critical care settings, particularly in the context of major
comorbidities of sepsis and burn trauma, such as ARDS [18,19] . Nephrilin has shown beneficial effects in
rodent models of sepsis and burn trauma, as noted above.

Finally, lung metastasis is accelerated by profibrotic and pro-metastatic factors such as CCL12 and
TIMP-1 [20,21] . We examined the possible effects of nephrilin on the circulating levels of these factors.

METHODS
Reagents
Nephrilin peptide (1) and FITC-labelled tracer peptide, the IGFBP3-derived portion of the nephrilin
peptide sequence (fitc-KKGFYKKKQCRPSKGRKRGFCW) were synthesized by Lifetein LLC
(Hillsborough, NJ). Antibodies for ELISAs were purchased from Abcam (Cambridge, MA), and chemicals
from Sigma-Aldrich (St. Louis, MO) unless otherwise specified.

Murine models
Animals were housed in clean cages on a 12 h light/dark cycle with access to standard chow and water
ad libitum. Animals were allowed to acclimate for one week before the experiment. All animal procedures
were performed in adherence to the National Institute of Health’s Guide for Care and Use of Laboratory