Polymeric nanoparticles alleviate myeloid cell-induced immunosuppression and inhibit metastasis in a material-dependent manner

Introduction: Metastasis is responsible for the majority of breast cancer-related deaths and depends on the formation of a metastatic niche at distal sites [1]. The metastatic niche consists primarily of immunosuppressive myeloid cells, such as neutrophils and monocytes. These cells develop aberrant phenotypes and are recruited to distal sites in response to cancer-induced immune dysregulation, promoting the recruitment and survival of disseminated tumor cells [2]. Depletion of cancer-associated myeloid cells has shown promise in treating metastasis in preclinical models, but has been limited by challenges with specificity and toxicity [3]. Novel therapies that specifically alter the phenotype and trafficking of myeloid cells may serve as a novel immunotherapy for cancer metastasis.

We have developed drug-free poly(lactide-co-glycolide) (PLG) nanoparticles to target myeloid cells and disrupt the formation of the metastatic niche. Nanoparticles are phagocytosed primarily by myeloid cells and alter the phenotype and trafficking of these cells. Polymeric nanoparticles have previously been used to ameliorate pathological inflammation in autoimmune disease, and were found to be safe and well-tolerated in clinical trials [4]. We found that PLG nanoparticles inhibited primary tumor growth and the formation of metastases, with efficacy dependent on nanoparticle composition. We investigated the mechanisms by which nanoparticle formulation influenced particle uptake, immune cell trafficking to the metastatic niche, and downstream effects on effector cell responses. We found that the nanoparticle surfactant influenced uptake by myeloid cells and resulting T cell phenotypes at the metastatic niche.


Materials and Methods: PLG nanoparticles were fabricated via a single-emulsion of 20% (w/v) PLG dissolved in dichloromethane with 2% poly(ethyl-alt-maleic) acid (PEMA) or 2% polyvinyl alcohol (PVA) (w/v) in water. For studies tracking nanoparticle association with cells, PLG conjugated with Cy5.5 via EDC-NHS chemistry was used to fabricate particles. Nanoparticles were washed, lyophilized, and analyzed with the Zetasizer for size and zeta potential. PLG nanoparticles were evaluated in the syngeneic 4T1 model of triple-negative breast cancer in female BALB/c or RAG1KO BALB/c mice. 100,000 4T1 cells labeled with luciferase were orthotopically inoculated into the fourth right mammary fat pad. Mice were intravenously injected with 1 mg of PEMA or PVA nanoparticles every 3 days starting 1 day after tumor inoculation. Lungs were extracted 14 or 21 days after inoculation for analysis of immune cell composition and phenotype by flow cytometry, single cell RNA-sequencing, or quantification of metastases by luminescent imaging.


Results and Discussion: Nanoparticles fabricated with PEMA or PVA had comparable sizes, with diameters of 619.3 ± 21.3 nm and 598.9 ± 47.7 nm, respectively. At this size scale, nanoparticles can be phagocytosed by myeloid cells. PEMA nanoparticles had a slightly more negative surface charge (-45.7 ± 2.2 mV) than PVA nanoparticles (-34.3 ± 0.9 mV), which may influence cell association. Both nanoparticle formulations significantly reduced primary tumor growth but varied in the inhibition of metastasis (Figure 1). Mice treated with PVA nanoparticles had 39.1% smaller tumors 21 days after inoculation compared to untreated mice, and a significantly reduced occurrence of lung metastasis (30% vs 90% in control mice). While mice treated with PEMA nanoparticles similarly had smaller tumors (32.6% reduction from control), 70% of these mice had metastases.

PEMA and PVA NPs had distinct effects on myeloid and T cell populations in the lung (Figure 2), which may explain differences in efficacy. PVA nanoparticles reduced the recruitment of adoptively transferred immune cells to the lung (51.4% decrease relative to untreated mice) and skewed the recruited cells towards Ly6C+ monocytes (204% more than untreated mice), while PEMA nanoparticles did not result in significant differences in recruitment. PVA nanoparticles accumulated at significantly higher rates in all lung cells and in dendritic cells than PEMA nanoparticles (75.6% and 519%, respectively. Dendritic cells, monocytes, and neutrophils in the lungs of PVA nanoparticle-treated mice had increased expression of genes associated with interferon and TNF signaling, as determined by single cell RNA sequencing. Mice treated with PVA nanoparticles had 161% more Tbet+ Th1 cells and 85.2% more RORγt+ Th17 cells at the lung than mice receiving PEMA nanoparticles, suggesting that nanoparticle-altered myeloid cell landscapes may influence T cell responses at the lung. Lastly, PVA nanoparticles failed to inhibit metastases in T and B cell deficient RAG1KO mice, indicating that effector cells are essential for nanoparticle efficacy (Figure 3).



Conclusion: Negatively charged PLG nanoparticles inhibited tumor growth and metastasis in an immunocompetent model of breast cancer. PVA nanoparticles, but not PEMA nanoparticles, significantly altered myeloid cell populations and T cell phenotypes at metastatic sites, and inhibited metastasis in a T-cell dependent manner. These findings suggest that cargo-free PLG nanoparticles could serve as a novel and tunable immunotherapy for treating cancer metastasis.


Acknowledgements (Optional): : Funding for this research was provided by the National Institutes of Health under award numbers R01CA243916 and R01CA214384.

References (Optional): : [1] Howlader N, Noone A, Krapcho M, Miller D, Brest A, Yu M, et al.. SEER cancer statistics review, 1975–2017. Natl Cancer Inst (2020) 4. Available at https://seer.cancer.gov/csr/1975_2017/. Based on November 2019 SEER data submission, posted to the SEER web site, April 2020.

[2] Swierczak A, Pollard JW. Myeloid cells in metastasis. Cold Spring Harb Perspect Med (2020) 10:a038026. doi: 10.1101/cshperspect.a038026

[3] Veglia F, Sanseviero E, Gabrilovich DI. Myeloid-derived suppressor cells in the era of increasing myeloid cell diversity. Nat Rev Immunol (2021) 21:485–98. doi: 10.1038/s41577-020-00490-y


[4] Kelly CP, Murray JA, Leffler DA, Getts DR, Bledsoe AC, Smithson G, First MR, Morris A, Boyne M, Elhofy A, Wu TT, Podojil JR, Miller SD; TAK-101 Study Group. TAK-101 Nanoparticles Induce Gluten-Specific Tolerance in Celiac Disease: A Randomized, Double-Blind, Placebo-Controlled Study. Gastroenterology. 2021 Jul;161(1):66-80.e8. doi: 10.1053/j.gastro.2021.03.014.