Although immune checkpoint blockade inhibitors (ICI) have shown convincing results in multiple cancers, the therapeutic efficacy is currently limited to 15–30% of treated cancer patients [
37]. Herein the new CXCR4 antagonist Pep R strengthen anti-PD-1 efficacy in two murine cancer models, MC38 colon cancer and B16-hCXCR4 melanoma, respectively reported to be immune responsive [
21] and immune resistant cancer models [
22,
24]. Increase in anti-PD-1 efficacy derived by TME modification potentiating the recruitment of Granzyme B positive and reducing Tregs cells. As previously reported [
33,
38], the increase of Granzyme B positive cells in MC38 tumors derived from mice treated with combined treatment, suggests that CXCR4 inhibition favors T effector access to TME. While Peptide R does not significantly increase the number of GRZB positive cells, it improves the anti-PD-1 efficacy toward a more infiltrated TME. CXCR4 inhibition favors T effector access to TME also in a more immune resistant model such as B16-hCXCR4. Pep R also reduced Treg infiltration in MC38 and B16-hCXCR4 tumors rendering the TME more immunoresponsive to anti-PD-1 therapy, as previously reported for B16 melanoma [
39]. CXCL12 and PD-L1 expression were reduced by Pep R treatment possibly through impairment of stromal/immuneregulatory cell recruitment [
12] and/ or transcriptional regulation [
40] while CXCR4 and PD-L1 expression were reduced in tumors treated with anti-PD-1 + Pep R. It was previously demonstrated that CXCR4 antagonists reshape TME favoring access of T effector and reducing the immunoregulatory cells in a model of pancreatic cancer [
11], hepatocellular carcinoma [
41] colorectal cancer [
14] and ovarian cancer [
13]. Potentiation in anti-CTLA-4 and anti-PD-1 efficacy was obtained with B16-GM-CSF expressing cell line (GVAX) [
35,
39]. Interestingly GM-CSF downregulated both CXCR4 and CXCL12 expression in bone marrow [
42]. In human metastatic triple negative breast cancer (TNBC) the dense fibrotic stroma is immunosuppressive and liver and lung metastases tend to be highly fibrotic excluding cytotoxic T lymphocytes (CTLs). Among genes that are associated with stromal T-lymphocyte exclusion there is CXCL12 [
43]. In a murine model of murine TNBC, the unique CXCR4 FDA approved antagonist, plerixafor, decreases fibrosis, increases CTL infiltration, and decreases immunosuppression doubling the response to immune checkpoint blockers [
43]. CXCR4 is overexpressed on Tregs, mainly the bone marrow retained Tregs, and CXCR4 peptide antagonist impairs Tregs function [
34]. Thus dual blockade of CXCL12-CXCR4 and PD-1-PD-L1 synergistically increases Teff/suppressive immune population in murine tumor models. Although not significant the combined treatment modified the content of myeloid-derived suppressor cells (MDSC) and plasmacytoid DC (pDC) in B16-hCXCR4. This can be explained by the TME-produced CXCL12 that is attractive not only for CXCR4+ Treg, but also for MDSC and pDC [
44‐
46]. It was reported that pDCs established an immunosuppressive TME impairing the response to TLR7/9 activation and decreasing IFN-α production [
47]. Moreover, genetic deletion of CXCR4 in myeloid cells (CXCR4MyeΔ/Δ) significantly reduced melanoma tumor growth enhancing the NK antitumor immune response. These data suggest that CXCR4-mediated signals from myeloid cells suppress NK cell–mediated tumor surveillance, and thereby enhance tumor growth [
48]. In terms of tumor growth, Pep R seems to confer a delay in tumor growth within the first week of treatment while a gain in growth is showed during the second week. Previously published data showed that Pep R reduced the growth of human renal cancer cells SN12C xenograft [
15] while Peptide S, although not impairing B16F10 tumor growth, reduced lung metastasis [
49]. In U87MG glioblastoma growth was unaffected by CXCR4 antagonists, AMD3100 and Pep R [
50]. Although not statistically significant this trend deserve further investigation. Most studies on PD-1 expression have focused on immune cells, rendering its potential expression and functions in tumor cells remaining largely unclear. To investigate the role of intrinsic PD-1 signaling in neoplastic cells, the effect of human anti-PD-1, nivolumab, was evaluated in combination with the newest and most powerful CXCR4 antagonist Pep R54 [
20] in a xenograft model of human melanoma. The combined effect of nivolumab + Pep R54 suggests a dual role for CXCR4 antagonism targeting tumoral cells and microenvironment. Herein, combined treatment of nivolumab + Pep R54 impaired tumor growth of human melanoma PES43 cells expressing PD-1 and CXCR4. In athymic mice, double targeting of CXCR4 and PD-1 significantly reduced human melanoma tumor growth as T cells independent effect. Targeting PD-1 and CXCR4 on PES43 melanoma cells reduced cell growth and inhibited survival signaling (pERK/pAkt) [
15‐
17] strengthen the effect of nivolumab that impaired pERK/pAkt and p4EBP1. We hypothesized that the CXCR4 antagonist Pep R54 plus anti-PD-1 simultaneously inhibits two core pathways of tumor proliferation, P-ERK/pAKT and p4EBP1.