Short-term immune-checkpoint inhibition partially rescues perturbed bone marrow hematopoiesis in mismatch-repair deficient tumors

ABSTRACT

Wide-spread cancer-related immunosuppression often curtails immune-mediated antitumoral responses. Immune-checkpoint inhibitors (ICIs) have become a state-of-the-art treatment modality for mismatch repair-deficient (dMMR) tumors. Still, the impact of ICI-treatment on bone marrow perturbations is largely unknown. Using anti-PD1 and anti-LAG-3 ICI treatments, we here investigated the effect of bone marrow hematopoiesis in tumor-bearing Msh2^{loxP/loxP;TgTg(Vil1-cre)} mice. The OS under anti-PD1 antibody treatment was 7.0 weeks (vs. 3.3 weeks and 5.0 weeks, control and isotype, respectively). In the anti-LAG-3 antibody group, OS was 13.3 weeks and thus even longer than in the anti-PD1 group (p = 0.13). Both ICIs induced a stable disease and reduced circulating and splenic regulatory T cells. In the bone marrow, a perturbed hematopoiesis was identified in tumor-bearing control mice, which was partially rescued by ICI treatment. In particular, B cell precursors and innate lymphoid progenitors were significantly increased upon anti-LAG-3 therapy to levels seen in tumor-free control mice. Additional normalizing effects of ICI treatment were observed for lin⁻c-Kit⁺IRF8⁺ hematopoietic stem cells, which function as a “master” negative regulator of the formation of polymorphonuclear-myeloid-derived suppressor cell generation. Accompanying immunofluorescence on the TME revealed significantly reduced numbers of CD206⁺F4/80⁺ and CD163⁺ tumor-associated M2 macrophages and CD11b⁺Gr1⁺ myeloid-derived suppressor cells especially upon anti-LAG-3 treatment. This study confirms the perturbed hematopoiesis in solid cancer. Anti-LAG-3 treatment partially restores normal hematopoiesis. The interference of anti-LAG-3 with suppressor cell populations in otherwise inaccessible niches renders this ICI very promising for subsequent clinical application.

GRAPHICAL ABSTRACT

Msh2^{loxP/loxP;TgTg(Vil1-cre)} mice develop tumors in the gastrointestinal tract because of a constitutional Msh2 knock out. Tumor growth is accompanied by global immunological changes in the peripheral blood, along with perturbations in the bone marrow and eventually spleen. These perturbations include a shifted hematopoiesis toward and unbalanced myelopoiesis. These immature cells are recruited to the tumor and polarized toward an immunosuppressive M2-like phenotype. This finally leads to tumor progression and global immunosuppression or exhaustion. Immune-checkpoint inhibitors may have the capacity to break this vicious circle via re-activation of the host’s immunity. Here, we could show that immune-checkpoint inhibition with anti-PD1 or anti-LAG-3 partially rebalances bone marrow perturbations via targeting of CSF-1R on hematopoietic progenitor cells. This suppressed recruitment of myeloid cells to the tumor and thus reshaped the tumor microenvironment. Finally, the outcome of tumor-bearing mice was significantly improved after short-term immune-checkpoint blockade.

KEYWORDS:

• Hematopoietic progenitor cell
• hypermutated tumor
• immunotherapy
• tumor microenvironment

Acknowledgments

We gratefully thank Mrs. Ilona Klamfuss and Ms. Chantal von Hoersten for breeding mice, Brigitte Vollmar and Bernd Krause for their continuous support in their efforts of chairing the Core Facility of Multimodal Small Animal Imaging. We also gratefully acknowledge the excellent technical assistance of Dr. Anna Schildt. Furthermore, we thank Carina Bergner and Anja Gummesson, radiopharmacy team of the Department of Nuclear Medicine of the University Medical Centre Rostock, for providing ¹⁸F-FDG for the small animal PET/CT experiments. Moreover, we thank Prof. Winfried Edelmann and Prof. Christoph Gasche for providing Msh2^{loxP/loxP;TgTg(Vil1-cre)} breeding pairs and giving the permission to breed mice in our facility.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Authors’ contributions

I.S. participated in in vivo experiments, isolated tumor RNA, and performed flow cytometry of blood, spleen, and bone marrow. P.K., L.E. J.T., J.M., and J.H. performed in vivo monitoring of health status and ex vivo analysis (including staining of blood and organs for flow cytometry), P.K. analyzed the bone marrow and isolated RNA from bone marrow, A.W. assisted in ex vivo analysis, including immunofluorescence staining and quantification, W.B. assisted in flow cytometry panel design, C.J. critically revised the manuscript, C.M. designed the study, analyzed data, prepared figures, and wrote the manuscript.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Ethics approval and consent to participate

The German local authority approved all animal experiments: Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei Mecklenburg‐Vorpommern (7221.3‐1‐062/19), under the German animal protection law and the EU Guideline 2010/63/EU. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/2162402X.2023.2230669

Additional information

Funding

This work was supported by grants from the German research foundation [DFG grant number MA5799/2-1 and MA5799/2-2] and the Brigitte und Dr. Konstanze Wegener-Stiftung to CM.