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Short-term starvation reduces IGF-1 levels to sensitize lung tumors to PD-1 immune checkpoint blockade

Abstract

Harnessing the immune system by blocking the programmed cell death protein 1 (PD-1) pathway has been a major breakthrough in non-small-cell lung cancer treatment. Nonetheless, many patients fail to respond to PD-1 inhibition. Using three syngeneic models, we demonstrate that short-term starvation synergizes with PD-1 blockade to inhibit lung cancer progression and metastasis. This antitumor activity was linked to a reduction in circulating insulin-like growth factor 1 (IGF-1) and a downregulation of IGF-1 receptor (IGF-1R) signaling in tumor cells. A combined inhibition of IGF-1R and PD-1 synergistically reduced tumor growth in mice. This effect required CD8 cells, boosted the intratumoral CD8/Treg ratio and led to the development of tumor-specific immunity. In patients with non-small-cell lung cancer, high plasma levels of IGF-1 or high IGF-1R expression in tumors was associated with resistance to anti-PD-1–programmed death-ligand 1 immunotherapy. In conclusion, our data strongly support the clinical evaluation of IGF-1 modulators in combination with PD-1 blockade.

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Fig. 1: STS in combination with PD-1 blockade has a therapeutic effect against lung cancer.
Fig. 2: STS in combination with PD-1 blockade increases the frequency of effector T cells within the tumor microenvironment.
Fig. 3: CD8 T cells are required for STS-induced sensitivity to anti-PD-1 blockade.
Fig. 4: The immunotherapeutic activity of STS + anti-PD-1 is mediated by a downregulation of the IGF-1–IGF-1R axis.
Fig. 5: Analysis of the downstream effectors of IGF-1 signaling in LLC cells under normal and STS-like conditions.
Fig. 6: The IGF-1–IGF-1R axis is associated with resistance to anti-PD-1–PD-L1 immunotherapy in patients with lung cancer.
Fig. 7: Combined PD-1–PD-L1 and IGF-1R blockade enhances tumor-specific immune response and synergistically protects against lung cancer.

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Data availability

Source data for Figs. 17 and Extended Data Figs. 17 are provided with the paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank C. Zandueta and O. Rogero for technical assistance, and J. M. Kurie (The University of Texas MD Anderson Cancer Center, Houston, TX) for gifting the 393P cells. This study was supported by the Foundation for Applied Medical Research (FIMA), CIBERONC (CB16/12/00443 and CB16/12/00364), Fundación Científica de la Asociación Española Contra el Cáncer, Fundación Ramón Areces, Juan Serrano, Instituto de Salud Carlos III–Fondo de Investigación Sanitaria–Fondo Europeo de Desarrollo Regional ‘Una manera de hacer Europa’ (FEDER; PI17/00411, PI16/01352, PI16/01821 and AC14/00034), La Caixa Foundation, Caja Navarra Foundation and the Spanish Ministry of Economy and Competitiveness (SAF2015-71606R, SAF2016-78568-R and RTI 2018-094507-B-100). F.E. is funded by a predoctoral fellowship from the Asociación de Amigos de la Universidad de Navarra and from La Caixa Foundation. S.O.-E. was funded by a predoctoral fellowship from the Asociación de Amigos de la Universidad de Navarra and is now supported by an FPU fellowship. M.F.S. is supported by a Miguel Servet type I contract from Instituto de Salud Carlos III–Fondo de Investigación Sanitaria.

Author information

Authors and Affiliations

Authors

Contributions

D.Ajona and R.P. supervised the study and were responsible for the study conception and design. J.M.L.-P., M.F.S., A.G., J.L.P.-G., I.G.-B., I.E.-S. and C.E.A. provided clinical samples. D.Ajona, S.O.-E., F.E., K.V., Y.S., S.V., C.S. and F.L. performed the in vivo studies. K.V. and C.B. prepared the viral vectors and the stable clones. D.Ajona, A.C. and M.R. were responsible of the Luminex analysis. D.Ajona and A.R. carried out the immunohistochemistry experiments. T.L. and J.J.L. performed the ELISpot assay. D.Alignani optimized and supervised the flow cytometry experiments. D.Ajona, I.M. C.B., A.R. and S.V. carried out the mRNA and protein expression analyses. D.Ajona, R.P., T.L., J.J.L., F.L., A.C., M.R., I.E.-S., M.F.S., C.E.A. and L.M.M. were responsible for the analysis and interpretation of the data. D.Ajona, M.F.S., I.E.-S. and R.P. performed the statistical analysis. D.Ajona and R.P. wrote the manuscript. All authors critically reviewed the manuscript and approved its final version.

Corresponding author

Correspondence to Daniel Ajona.

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Extended data

Extended Data Fig. 1 STS/anti-PD-1 treatment impairs 393P and LLC tumor growth.

a, Individual follow-up of 393P tumor volumes in mice treated with anti-PD-1 (n=10 mice), STS (n=10 mice), the combination of both (n=8 mice) or vehicle (Control; n=11 mice). b, Individual follow-up of LLC tumor volumes in mice treated with anti-PD-1 (n=8 mice), STS (n=6 mice), the combination of both (n=6 mice) or vehicle (Control; n=8 mice). Numerical source data are provided as a source data file.

Source data

Extended Data Fig. 2 Effects of STS/anti-PD-1 combination in LLC tumor immune infiltrate.

a, Flow cytometric analysis of the proportion of intratumoral CD4, NK (NK1.1) and B (CD19) cells at day 18 after subcutaneous inoculation of LLC cells (Control, n=8 tumors; anti-PD-1, n=8 tumors; STS, n=5 tumors; STS/anti-PD-1, n=7 tumors). b, Expression of PD-1 in tumor-infiltrating CD4 T cells of the experiment shown in section a. Data are expressed as the mean fluorescence intensity (MFI) ± s.e.m. c, Flow cytometric analysis of the proportion of intratumoral total MDSCs, monocytic MDSCs (M-MDSCs; CD11b/Ly6C+/Ly6Glow), granulocytic MDSCs (G-MDSC; CD11b/Ly6C+/Ly6G+), macrophages (F4/80) and DCs (CD11c). The number of tumors per group was: Control, n=7 tumors; anti-PD-1, n=8 tumors; STS, n=5 tumors; STS/anti-PD-1 combination, n=7 tumors. Data are expressed as the mean of the percentage of total leukocytes (CD45) ± s.e.m. In all cases, the statistical significance of the differences were evaluated using the two-sided Kruskal-Wallis test with the Mann-Whitney U-test as the post hoc test. Numerical source data are provided as a source data file.

Source data

Extended Data Fig. 3 Effects of STS/anti-PD-1 combination in the proportion of immune circulating cells from LLC tumor-bearing mice.

Flow cytometric analysis of the proportion of peripheral blood CD8, CD4, Treg cells, NK (NK1.1) and B (CD19) cells in mice after inoculation of LLC cells and two cycles of 48 hours of STS (day 12 after tumor implantation) (Control, n=6 mice; anti-PD-1, n=6 mice; STS, n=5 mice; STS/anti-PD-1, n=6 mice). Data are expressed as the percentage of total lymphocytes except for Treg cells, which are expressed as the percentage of total CD4 T cells (means ± s.e.m.). The differences between experimental groups were analyzed using the two-sided Kruskal-Wallis test with the Mann-Whitney U-test as the post hoc test. Numerical source data are provided as a source data file.

Source data

Extended Data Fig. 4 IGF-1 inhibits the increase in the LC3-II/LC3-I ratio induced by in vitro STS.

a, Cropping images from the western blot analysis of LC3-I and LC3-II in LLC cells cultured under normal or STS-like conditions for 48 hours. This experiment was performed twice with similar results. b, Cropping images from the western blot analysis of LC3-I and LC3-II in LLC cells cultured under normal or STS-like conditions in the presence of 1 µg ml–1 of rIGF-1 or vehicle for 48 and 72 hours. This experiment was performed once. Unprocessed images of blots are provided as source data file.

Source data

Extended Data Fig. 5 PQ401/anti-PD-1 treatment impairs 393P tumor growth.

Individual follow-up of 393P tumor volumes in mice treated with PQ401 (n=10 mice), anti-PD-1 (n=11 mice), or the combination of both (n=10 mice) or vehicle (Control; n=11 mice). Numerical source data are provided as a source data file.

Source data

Extended Data Fig. 6 Effects of PQ401/anti-PD-1 combination in LLC tumor immune infiltrate.

a, Flow cytometric analysis of the proportion of intratumoral total MDSCs, monocytic MDSCs (M-MDSCs; CD11b/Ly6C+/Ly6Glow), granulocytic MDSCs (G-MDSC; CD11b/Ly6C+/Ly6G+), macrophages (F4/80), DCs (CD11c), CD4 T, NK (NK1.1) and B (CD19) cells at day 19 after inoculation of LLC cells (n=8 tumors per group except for anti-PD-1 group in CD4 T, NK and B cells analyses; n=7). Data are expressed as the percentage of total leukocytes (CD45). b, Expression of the markers PD-1, GITR, and LAG-3 in tumor-infiltrating CD4 T cells. The number of tumors per group was: Control, n=8 tumors; anti-PD-1, n=7 tumors; PQ401, n=8 tumors; PQ401/anti-PD-1 combination, n=8 tumors. Data are expressed as the mean of fluorescence intensity (MFI) ± s.e.m. The differences between experimental groups were analyzed using the two-sided Kruskal-Wallis test with the Mann-Whitney U-test as the post hoc test. Numerical source data are provided as a source data file.

Source data

Extended Data Fig. 7 Effects of PQ401/anti-PD-1 combination in splenic immune populations from LLC tumor-bearing mice.

Flow cytometric analysis of the proportion of splenic CD8, CD4, B cells (CD19), NK (NK1.1) and Treg cells, MDSCs (total MDSCs, M-MDSCs, and G-MDSCs), macrophages, and DCs in tumor-bearing mice at day 19 after inoculation of LLC cells (n=8 mice per group). Data are expressed as the percentage of total leukocytes (CD45), except for Treg cells, which are expressed as the percentage of total CD4 T cells (means ± s.e.m.). The differences between experimental groups were analyzed using the two-sided Kruskal-Wallis test with the Mann-Whitney U-test as the post hoc test. No statistically significant differences were found. Numerical source data are provided as a source data file.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2 and Supplementary Figs. 1 and 2.

Reporting Summary

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Ajona, D., Ortiz-Espinosa, S., Lozano, T. et al. Short-term starvation reduces IGF-1 levels to sensitize lung tumors to PD-1 immune checkpoint blockade. Nat Cancer 1, 75–85 (2020). https://doi.org/10.1038/s43018-019-0007-9

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