Targeting CXCR4 potentiates anti-PD-1 efficacy modifying the tumor microenvironment and inhibiting neoplastic PD-1

Crescenzo D'Alterio, Maria Buoncervello, Caterina Ieranò, Maria Napolitano, Luigi Portella, Giuseppina Rea, Antonio Barbieri, Antonio Luciano, Giosuè Scognamiglio, Fabiana Tatangelo, Anna Maria Anniciello, Mario Monaco, Ernesta Cavalcanti, Piera Maiolino, Giulia Romagnoli, Claudio Arra, Gerardo Botti, Lucia Gabriele, Stefania Scala

Research output: Contribution to journalArticle

Abstract

Background: Inefficient T-cell access to the tumor microenvironment (TME) is among the causes of tumor immune-resistance. Previous evidence demonstrated that targeting CXCR4 improves anti-PD-1/PD-L1 efficacy reshaping TME. To evaluate the role of newly developed CXCR4 antagonists (PCT/IB2011/000120/ EP2528936B1/US2013/0079292A1) in potentiating anti-PD-1 efficacy two syngeneic murine models, the MC38 colon cancer and the B16 melanoma-human CXCR4-transduced, were employed. Methods: Mice were subcutaneously injected with MC38 (1 × 106) or B16-hCXCR4 (5 × 105). After two weeks, tumors bearing mice were intraperitoneally (ip) treated with murine anti-PD-1 [RMP1-14] (5 mg/kg, twice week for 2 weeks), Pep R (2 mg/kg, 5 days per week for 2 weeks), or both agents. The TME was evaluated through immunohistochemistry and flow-cytometry. In addition, the effects of the human-anti-PD-1 nivolumab and/or Peptide-R54 (Pep R54), were evaluated on human melanoma PES43 cells and xenografts treated. Results: The combined treatment, Pep R plus anti-PD-1, reduced the MC38 Relative Tumor Volume (RTV) by 2.67 fold (p = 0.038) while nor anti-PD-1, neither Pep R significantly impacted on tumor growth. Significant higher number of Granzyme B (GZMB) positive cells was detected in MC38 tumors from mice treated with the combined treatment (p = 0.016) while anti-PD-1 determined a modest but significant increase of tumor-infiltrating GZMB positive cells (p = 0.035). Also, a lower number of FoxP3 positive cells was detected (p = 0.022). In the B16-hCXCR4 tumors, two weeks of combined treatment reduced tumor volume by 2.27 fold while nor anti-PD-1 neither Pep R significantly impacted on tumor growth. A significant higher number of GRZB positive cells was observed in B16-hCXCR4 tumors treated with combined treatment (p = 0,0015) as compared to anti-PD-1 (p = 0.028). The combined treatment reduced CXCR4, CXCL12 and PD-L1 expression in MC38 tumors. In addition, flow cytometry on fresh B16-hCXCR4 tumors showed significantly higher Tregs number following anti-PD-1 partially reversed by the combined treatment Pep R and anti-PD-1. Combined treatment determined an increase of CD8/Tregs and CD8/MDSC ratio. To dissect the effect of anti-PD-1 and CXCR4 targeting on PD-1 expressed by human cancer cells, PES43 human melanoma xenograft model was employed. In vitro human anti-PD-1 nivolumab or pembrolizumab (10 μM) reduced PES43 cells growth while nivolumab (10 μM) inhibited pERK1/2, P38 MAPK, pAKT and p4EBP. PES43 xenograft mice were treated with Pep R54, a newly developed Pep R derivative (AcHN-Arg-Ala-[DCys-Arg- Nal(2′)-His-Pen]- COOH), plus nivolumab. After 3 weeks of combined treatment a significant reduction in tumor growth was shown (p = 0.038). PES43 lung disseminated tumor cells (DTC) were detected in fresh lung tissues as melanoma positive MCSP-APC+ cells. Although not statistically significant, DTC-PES43 cells were reduced in mice lungs treated with combined treatment while nivolumab or Pep R54 did not affect DTC number. Conclusion: Combined treatment with the new developed CXCR4 antagonist, Pep R, plus anti-PD-1, reduced tumor-growth in two syngeneic murine models, anti-PD-1 sensitive and resistant, potentiating Granzyme and reducing Foxp3 cells infiltration. In addition, the human specific CXCR4 antagonist, Pep R54, cooperated with nivolumab in inhibiting the growth of the PD-1 expressing human PES43 melanoma xenograft. This evidence sheds light on PD-1 targeting mechanisms and paves the way for CXCR4/PD-1 targeting combination therapy.

Original languageEnglish
Article number432
JournalJournal of Experimental and Clinical Cancer Research
Volume38
Issue number1
DOIs
Publication statusPublished - Oct 28 2019

Fingerprint

Tumor Microenvironment
Neoplasms
Heterografts
Granzymes
Melanoma
Growth
Peptides
Therapeutics
Tumor Burden
Lung
Flow Cytometry
Experimental Melanomas
p38 Mitogen-Activated Protein Kinases

Keywords

  • Immune privilege
  • MDSC; CXCR4-CXCL12 pathway
  • Treg
  • Tumor infiltrating lymphocytes
  • Tumor intrinsic PD-1 pathway
  • Tumor microenvironment

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

@article{e7d68eccecbd4186a6ca882be062a8ab,
title = "Targeting CXCR4 potentiates anti-PD-1 efficacy modifying the tumor microenvironment and inhibiting neoplastic PD-1",
abstract = "Background: Inefficient T-cell access to the tumor microenvironment (TME) is among the causes of tumor immune-resistance. Previous evidence demonstrated that targeting CXCR4 improves anti-PD-1/PD-L1 efficacy reshaping TME. To evaluate the role of newly developed CXCR4 antagonists (PCT/IB2011/000120/ EP2528936B1/US2013/0079292A1) in potentiating anti-PD-1 efficacy two syngeneic murine models, the MC38 colon cancer and the B16 melanoma-human CXCR4-transduced, were employed. Methods: Mice were subcutaneously injected with MC38 (1 × 106) or B16-hCXCR4 (5 × 105). After two weeks, tumors bearing mice were intraperitoneally (ip) treated with murine anti-PD-1 [RMP1-14] (5 mg/kg, twice week for 2 weeks), Pep R (2 mg/kg, 5 days per week for 2 weeks), or both agents. The TME was evaluated through immunohistochemistry and flow-cytometry. In addition, the effects of the human-anti-PD-1 nivolumab and/or Peptide-R54 (Pep R54), were evaluated on human melanoma PES43 cells and xenografts treated. Results: The combined treatment, Pep R plus anti-PD-1, reduced the MC38 Relative Tumor Volume (RTV) by 2.67 fold (p = 0.038) while nor anti-PD-1, neither Pep R significantly impacted on tumor growth. Significant higher number of Granzyme B (GZMB) positive cells was detected in MC38 tumors from mice treated with the combined treatment (p = 0.016) while anti-PD-1 determined a modest but significant increase of tumor-infiltrating GZMB positive cells (p = 0.035). Also, a lower number of FoxP3 positive cells was detected (p = 0.022). In the B16-hCXCR4 tumors, two weeks of combined treatment reduced tumor volume by 2.27 fold while nor anti-PD-1 neither Pep R significantly impacted on tumor growth. A significant higher number of GRZB positive cells was observed in B16-hCXCR4 tumors treated with combined treatment (p = 0,0015) as compared to anti-PD-1 (p = 0.028). The combined treatment reduced CXCR4, CXCL12 and PD-L1 expression in MC38 tumors. In addition, flow cytometry on fresh B16-hCXCR4 tumors showed significantly higher Tregs number following anti-PD-1 partially reversed by the combined treatment Pep R and anti-PD-1. Combined treatment determined an increase of CD8/Tregs and CD8/MDSC ratio. To dissect the effect of anti-PD-1 and CXCR4 targeting on PD-1 expressed by human cancer cells, PES43 human melanoma xenograft model was employed. In vitro human anti-PD-1 nivolumab or pembrolizumab (10 μM) reduced PES43 cells growth while nivolumab (10 μM) inhibited pERK1/2, P38 MAPK, pAKT and p4EBP. PES43 xenograft mice were treated with Pep R54, a newly developed Pep R derivative (AcHN-Arg-Ala-[DCys-Arg- Nal(2′)-His-Pen]- COOH), plus nivolumab. After 3 weeks of combined treatment a significant reduction in tumor growth was shown (p = 0.038). PES43 lung disseminated tumor cells (DTC) were detected in fresh lung tissues as melanoma positive MCSP-APC+ cells. Although not statistically significant, DTC-PES43 cells were reduced in mice lungs treated with combined treatment while nivolumab or Pep R54 did not affect DTC number. Conclusion: Combined treatment with the new developed CXCR4 antagonist, Pep R, plus anti-PD-1, reduced tumor-growth in two syngeneic murine models, anti-PD-1 sensitive and resistant, potentiating Granzyme and reducing Foxp3 cells infiltration. In addition, the human specific CXCR4 antagonist, Pep R54, cooperated with nivolumab in inhibiting the growth of the PD-1 expressing human PES43 melanoma xenograft. This evidence sheds light on PD-1 targeting mechanisms and paves the way for CXCR4/PD-1 targeting combination therapy.",
keywords = "Immune privilege, MDSC; CXCR4-CXCL12 pathway, Treg, Tumor infiltrating lymphocytes, Tumor intrinsic PD-1 pathway, Tumor microenvironment",
author = "Crescenzo D'Alterio and Maria Buoncervello and Caterina Ieran{\`o} and Maria Napolitano and Luigi Portella and Giuseppina Rea and Antonio Barbieri and Antonio Luciano and Giosu{\`e} Scognamiglio and Fabiana Tatangelo and Anniciello, {Anna Maria} and Mario Monaco and Ernesta Cavalcanti and Piera Maiolino and Giulia Romagnoli and Claudio Arra and Gerardo Botti and Lucia Gabriele and Stefania Scala",
year = "2019",
month = "10",
day = "28",
doi = "10.1186/s13046-019-1420-8",
language = "English",
volume = "38",
journal = "Journal of Experimental and Clinical Cancer Research",
issn = "0392-9078",
publisher = "BioMed Central Ltd.",
number = "1",

}

TY - JOUR

T1 - Targeting CXCR4 potentiates anti-PD-1 efficacy modifying the tumor microenvironment and inhibiting neoplastic PD-1

AU - D'Alterio, Crescenzo

AU - Buoncervello, Maria

AU - Ieranò, Caterina

AU - Napolitano, Maria

AU - Portella, Luigi

AU - Rea, Giuseppina

AU - Barbieri, Antonio

AU - Luciano, Antonio

AU - Scognamiglio, Giosuè

AU - Tatangelo, Fabiana

AU - Anniciello, Anna Maria

AU - Monaco, Mario

AU - Cavalcanti, Ernesta

AU - Maiolino, Piera

AU - Romagnoli, Giulia

AU - Arra, Claudio

AU - Botti, Gerardo

AU - Gabriele, Lucia

AU - Scala, Stefania

PY - 2019/10/28

Y1 - 2019/10/28

N2 - Background: Inefficient T-cell access to the tumor microenvironment (TME) is among the causes of tumor immune-resistance. Previous evidence demonstrated that targeting CXCR4 improves anti-PD-1/PD-L1 efficacy reshaping TME. To evaluate the role of newly developed CXCR4 antagonists (PCT/IB2011/000120/ EP2528936B1/US2013/0079292A1) in potentiating anti-PD-1 efficacy two syngeneic murine models, the MC38 colon cancer and the B16 melanoma-human CXCR4-transduced, were employed. Methods: Mice were subcutaneously injected with MC38 (1 × 106) or B16-hCXCR4 (5 × 105). After two weeks, tumors bearing mice were intraperitoneally (ip) treated with murine anti-PD-1 [RMP1-14] (5 mg/kg, twice week for 2 weeks), Pep R (2 mg/kg, 5 days per week for 2 weeks), or both agents. The TME was evaluated through immunohistochemistry and flow-cytometry. In addition, the effects of the human-anti-PD-1 nivolumab and/or Peptide-R54 (Pep R54), were evaluated on human melanoma PES43 cells and xenografts treated. Results: The combined treatment, Pep R plus anti-PD-1, reduced the MC38 Relative Tumor Volume (RTV) by 2.67 fold (p = 0.038) while nor anti-PD-1, neither Pep R significantly impacted on tumor growth. Significant higher number of Granzyme B (GZMB) positive cells was detected in MC38 tumors from mice treated with the combined treatment (p = 0.016) while anti-PD-1 determined a modest but significant increase of tumor-infiltrating GZMB positive cells (p = 0.035). Also, a lower number of FoxP3 positive cells was detected (p = 0.022). In the B16-hCXCR4 tumors, two weeks of combined treatment reduced tumor volume by 2.27 fold while nor anti-PD-1 neither Pep R significantly impacted on tumor growth. A significant higher number of GRZB positive cells was observed in B16-hCXCR4 tumors treated with combined treatment (p = 0,0015) as compared to anti-PD-1 (p = 0.028). The combined treatment reduced CXCR4, CXCL12 and PD-L1 expression in MC38 tumors. In addition, flow cytometry on fresh B16-hCXCR4 tumors showed significantly higher Tregs number following anti-PD-1 partially reversed by the combined treatment Pep R and anti-PD-1. Combined treatment determined an increase of CD8/Tregs and CD8/MDSC ratio. To dissect the effect of anti-PD-1 and CXCR4 targeting on PD-1 expressed by human cancer cells, PES43 human melanoma xenograft model was employed. In vitro human anti-PD-1 nivolumab or pembrolizumab (10 μM) reduced PES43 cells growth while nivolumab (10 μM) inhibited pERK1/2, P38 MAPK, pAKT and p4EBP. PES43 xenograft mice were treated with Pep R54, a newly developed Pep R derivative (AcHN-Arg-Ala-[DCys-Arg- Nal(2′)-His-Pen]- COOH), plus nivolumab. After 3 weeks of combined treatment a significant reduction in tumor growth was shown (p = 0.038). PES43 lung disseminated tumor cells (DTC) were detected in fresh lung tissues as melanoma positive MCSP-APC+ cells. Although not statistically significant, DTC-PES43 cells were reduced in mice lungs treated with combined treatment while nivolumab or Pep R54 did not affect DTC number. Conclusion: Combined treatment with the new developed CXCR4 antagonist, Pep R, plus anti-PD-1, reduced tumor-growth in two syngeneic murine models, anti-PD-1 sensitive and resistant, potentiating Granzyme and reducing Foxp3 cells infiltration. In addition, the human specific CXCR4 antagonist, Pep R54, cooperated with nivolumab in inhibiting the growth of the PD-1 expressing human PES43 melanoma xenograft. This evidence sheds light on PD-1 targeting mechanisms and paves the way for CXCR4/PD-1 targeting combination therapy.

AB - Background: Inefficient T-cell access to the tumor microenvironment (TME) is among the causes of tumor immune-resistance. Previous evidence demonstrated that targeting CXCR4 improves anti-PD-1/PD-L1 efficacy reshaping TME. To evaluate the role of newly developed CXCR4 antagonists (PCT/IB2011/000120/ EP2528936B1/US2013/0079292A1) in potentiating anti-PD-1 efficacy two syngeneic murine models, the MC38 colon cancer and the B16 melanoma-human CXCR4-transduced, were employed. Methods: Mice were subcutaneously injected with MC38 (1 × 106) or B16-hCXCR4 (5 × 105). After two weeks, tumors bearing mice were intraperitoneally (ip) treated with murine anti-PD-1 [RMP1-14] (5 mg/kg, twice week for 2 weeks), Pep R (2 mg/kg, 5 days per week for 2 weeks), or both agents. The TME was evaluated through immunohistochemistry and flow-cytometry. In addition, the effects of the human-anti-PD-1 nivolumab and/or Peptide-R54 (Pep R54), were evaluated on human melanoma PES43 cells and xenografts treated. Results: The combined treatment, Pep R plus anti-PD-1, reduced the MC38 Relative Tumor Volume (RTV) by 2.67 fold (p = 0.038) while nor anti-PD-1, neither Pep R significantly impacted on tumor growth. Significant higher number of Granzyme B (GZMB) positive cells was detected in MC38 tumors from mice treated with the combined treatment (p = 0.016) while anti-PD-1 determined a modest but significant increase of tumor-infiltrating GZMB positive cells (p = 0.035). Also, a lower number of FoxP3 positive cells was detected (p = 0.022). In the B16-hCXCR4 tumors, two weeks of combined treatment reduced tumor volume by 2.27 fold while nor anti-PD-1 neither Pep R significantly impacted on tumor growth. A significant higher number of GRZB positive cells was observed in B16-hCXCR4 tumors treated with combined treatment (p = 0,0015) as compared to anti-PD-1 (p = 0.028). The combined treatment reduced CXCR4, CXCL12 and PD-L1 expression in MC38 tumors. In addition, flow cytometry on fresh B16-hCXCR4 tumors showed significantly higher Tregs number following anti-PD-1 partially reversed by the combined treatment Pep R and anti-PD-1. Combined treatment determined an increase of CD8/Tregs and CD8/MDSC ratio. To dissect the effect of anti-PD-1 and CXCR4 targeting on PD-1 expressed by human cancer cells, PES43 human melanoma xenograft model was employed. In vitro human anti-PD-1 nivolumab or pembrolizumab (10 μM) reduced PES43 cells growth while nivolumab (10 μM) inhibited pERK1/2, P38 MAPK, pAKT and p4EBP. PES43 xenograft mice were treated with Pep R54, a newly developed Pep R derivative (AcHN-Arg-Ala-[DCys-Arg- Nal(2′)-His-Pen]- COOH), plus nivolumab. After 3 weeks of combined treatment a significant reduction in tumor growth was shown (p = 0.038). PES43 lung disseminated tumor cells (DTC) were detected in fresh lung tissues as melanoma positive MCSP-APC+ cells. Although not statistically significant, DTC-PES43 cells were reduced in mice lungs treated with combined treatment while nivolumab or Pep R54 did not affect DTC number. Conclusion: Combined treatment with the new developed CXCR4 antagonist, Pep R, plus anti-PD-1, reduced tumor-growth in two syngeneic murine models, anti-PD-1 sensitive and resistant, potentiating Granzyme and reducing Foxp3 cells infiltration. In addition, the human specific CXCR4 antagonist, Pep R54, cooperated with nivolumab in inhibiting the growth of the PD-1 expressing human PES43 melanoma xenograft. This evidence sheds light on PD-1 targeting mechanisms and paves the way for CXCR4/PD-1 targeting combination therapy.

KW - Immune privilege

KW - MDSC; CXCR4-CXCL12 pathway

KW - Treg

KW - Tumor infiltrating lymphocytes

KW - Tumor intrinsic PD-1 pathway

KW - Tumor microenvironment

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U2 - 10.1186/s13046-019-1420-8

DO - 10.1186/s13046-019-1420-8

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