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Neutrophils bearing adhesive polymer micropatches as a drug-free cancer immunotherapy

Nature Biomedical Engineering (2024)Cite this article

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Abstract

Tumour-associated neutrophils can exert antitumour effects but can also assume a pro-tumoural phenotype in the immunosuppressive tumour microenvironment. Here we show that neutrophils can be polarized towards the antitumour phenotype by discoidal polymer micrometric ‘patches’ that adhere to the neutrophils’ surfaces without being internalized. Intravenously administered micropatch-loaded neutrophils accumulated in the spleen and in tumour-draining lymph nodes, and activated splenic natural killer cells and T cells, increasing the accumulation of dendritic cells and natural killer cells. In mice bearing subcutaneous B16F10 tumours or orthotopic 4T1 tumours, intravenous injection of the micropatch-loaded neutrophils led to robust systemic immune responses, a reduction in tumour burden and improvements in survival rates. Micropatch-activated neutrophils combined with the checkpoint inhibitor anti-cytotoxic T-lymphocyte-associated protein 4 resulted in strong inhibition of the growth of B16F10 tumours, and in complete tumour regression in one-third of the treated mice. Micropatch-loaded neutrophils could provide a potent, scalable and drug-free approach for neutrophil-based cancer immunotherapy.

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Fig. 1: CAMPs can be efficiently attached to neutrophils.
Fig. 2: CAMP attachment leads to activation of neutrophils.
Fig. 3: Treatment with CAMPs altered the transcriptomic profile of neutrophils.
Fig. 4: Biodistribution of neutrophils in B16F10-tumour-bearing mice.
Fig. 5: Treatment with neutrophil–CAMPs enhances systemic immune response.
Fig. 6: Systemic dose of CAMP-carrying neutrophils reduces tumour burden.

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Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. Raw bulk RNA-seq data for Fig. 3 and Extended Data Fig. 1 are available from NCBI Sequence Read Archive under accession number PRJNA1067075. Source data for tumour progression and survival data from Fig. 6 and Extended Data Figs. 4 and 5 are provided with this paper. All raw and analysed datasets generated during the study are available from the corresponding author on reasonable request.

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Acknowledgements

We acknowledge R. Bronson of Rodent Histopathology Core at the Harvard Medical School for histological evaluation. We acknowledge the use of BioRender.com for schematic creation. We acknowledge the Harvard Center for Biological Imaging; the Allston Science and Engineering Complex’s Molecular and Cellular Biology Core; and the Harvard University Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Coordinated Infrastructure Network (NNCI) supported by the National Science Foundation ECCS-2025158. Bulk RNA-seq was performed by Bauer Sequencing Core at Harvard University. We acknowledge funding from the John A. Paulson School of Engineering and Applied Sciences, and Wyss Institute for Biologically Inspired Engineering. N. Kapate was supported by the National Science Foundation Graduate Research Fellowship under grant number 1122374.

Author information

Author notes

  1. Tatsuya Fukuta

    Present address: Department of Physical Pharmaceutics, School of Pharmaceutical Sciences, Wakayama Medical University, Wakayama, Japan

  2. Rocky An

    Present address: Department of Bioengineering, Stanford University, Stanford, CA, USA

  3. Alexander P. Gottlieb

    Present address: Division of Breast Surgery, Department of Surgery, Brigham and Women’s Hospital, Boston, MA, USA

Authors and Affiliations

  1. Harvard John A. Paulson School of Engineering and Applied Sciences, Harvard University, Allston, MA, USA

    Ninad Kumbhojkar, Supriya Prakash, Tatsuya Fukuta, Neha Kapate, Rocky An, Solomina Darko, Vineeth Chandran Suja, Kyung Soo Park, Alexander P. Gottlieb, Michael Griffith Bibbey, Lily Li-Wen Wang, David J. Mooney & Samir Mitragotri

  2. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA

    Ninad Kumbhojkar, Supriya Prakash, Tatsuya Fukuta, Kwasi Adu-Berchie, Neha Kapate, Vineeth Chandran Suja, Kyung Soo Park, Alexander P. Gottlieb, Michael Griffith Bibbey, Malini Mukherji, Lily Li-Wen Wang, David J. Mooney & Samir Mitragotri

  3. Harvard–MIT Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA

    Neha Kapate & Lily Li-Wen Wang

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Contributions

N. Kumbhojkar and S.M. conceived the project and designed the experiments. N. Kumbhojkar, S.P., T.F., N. Kapate, R.A., S.D., V.C.S., K.S.P., A.P.G., M.M., M.G.B. and L.L.-W.W. performed the experiments. K.A.-B. and D.J.M designed and performed the RNA-seq data analysis. N. Kumbhojkar analysed data with co-authors. N. Kumbhojkar and S.M. wrote the paper with input from all the co-authors.

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Correspondence to Samir Mitragotri.

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Competing interests

N. Kumbhojkar, S.P., N. Kapate, L.L.-W.W. and S.M. are inventors of patent applications related to the technologies discussed in this work. These patents are owned and managed by Harvard University. S.M. declares the following interests: Hitch Bio, board member, equity; and Asalyxa, scientific advisory board (SAB), equity. D.J.M. declares the following competing interests: Novartis, sponsored research, licensed intellectual property; Immulus, equity; IVIVA, SAB; Attivare, SAB, equity; and Lyell, licensed intellectual property, equity. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 The transcriptomic profile of the CAMP-treated neutrophils is altered.

a, Volcano plot showing over 2000 differentially expressed genes between CAMP -ve NE and NE. Threshold P = 0.01. b, Significantly overexpressed pathways in CAMP -ve NE when compared to NE. Generated using EnrichR, focusing on MSigDB Hallmark 2020 database. c-d, Violin plots comparing aggregate expression distribution of genes related to IL-1 pathway (c) and IL-6-JAK-STAT3 pathway (d) according to MSigDB Hallmark 2020 database. Adhesion to CAMPs led to the increased aggregate expression of genes related to IL-1 and IL-6-JAK-STAT3 pathways. e-f, Violin Plots representing the expression of N2 related genes Arg-1 (e) and CD206 (f). Adhesion to CAMPs led to reduction in the expression of N2 related genes. Solid line within each violin represents median and dotted lines represent quartiles. Analysed using ordinary one-way ANOVA with Tukey’s multiple comparisons test.

Extended Data Fig. 2 Biodistribution of adoptively transferred neutrophils.

a, Experimental scheme for biodistribution in the B16F10 model. b, Representative IVIS images for organ accumulation of neutrophils in the B16F10 model. c, Experimental scheme for biodistribution in the 4T1-Luc model. d-f Biodistribution of untreated neutrophils (NE) and neutrophil-CAMPs (NE + CAMP) in the 4T1-Luc model at 24 (d), 48 (e) and 72 (f) hours after intravenous injections. n = 3 biological replicates. Data represented as mean ± SD. Adoptively transferred CAMP-treated neutrophils efficiently accumulated in the tumour, spleen, lungs, and liver of 4T1-Luc tumour bearing mice. g, IVIS images for tumour accumulation of neutrophils in the 4T1-Luc model at different timepoints. h, Kinetics of adoptively transferred neutrophils in the blood circulation as estimated by flow cytometry. Y axis represents %vivotrack680+ neutrophils of all neutrophils in circulation. n = 3 biological replicates. Data represented as mean ± SEM. Analysed by ordinary two-way ANOVA with Sidak’s multiple comparisons tests.

Extended Data Fig. 3 Treatment with CAMP-carrying neutrophils elicits a systemic immune response in the B16F10 model.

a, Experimental scheme for immunophenotyping. B16F10-tumour-bearing mice were treated with two doses of Saline, Neutrophil only (NE) or neutrophil-CAMPs (NE + CAMP). Blood, tumours, tumour-draining lymph nodes and spleens were harvested 48 hours after the second dose and analysed using flow cytometry. b, Neutrophil (%Ly6G+ Ly6C+ of CD45+) accumulation in the spleen was higher in the NE + CAMP group. c, Neutrophil accumulation in the tumour-draining lymph nodes was higher in the NE + CAMP group. d, CD8 + T cells in the spleen displayed higher expression of PD-1 in NE + CAMP group. e-f, NK cells (e) and CD8 + T cells (f) in the spleen displayed higher expression of LAMP-1 in the NE + CAMP group. g, The proportion of CD8+ effector memory T cells in the spleen was reduced in the NE + CAMP group. h, The proportion of CD8+ effector memory T cells in the tumour was marginally increased in the NE + CAMP group. i, CD8 + T cells in the blood in the NE + CAMP groups displayed activated phenotype as seen from the higher expression of PD-1. j-k, CD8 + T cells (j) and NK cells (k) in the tumours in the NE + CAMP group displayed modest activation with a marginally higher expression of CD69. l, Marginally higher accumulation of neutrophils in the tumours was observed in NE + CAMP group. n = 6 biological replicates for saline, n = 8 biological replicates for NE, n = 8 biological replicates for NE + CAMP. All data are represented as mean ± SEM. Sentences at the top of each panel represent the key observation that pertains to the NE + CAMP group. Data analysed using Ordinary one-way ANOVA with Tukey’s multiple comparisons test.

Extended Data Fig. 4 Systemic dose of CAMP-carrying neutrophils reduces tumor burden in 4T1-Luc model.

a, Experimental scheme for the treatment of 4T1-Luc tumours. Mice bearing orthotopic 4T1-Luc tumours were dosed twice with saline, neutrophils alone (NE) or neutrophil-CAMPs (NE + CAMP). First dose was performed when the tumours reached an average size of 50 mm3. b, Tumour growth kinetics of various treatments in the 4T1-Luc model. Neutrophil-CAMPs treatment significantly slowed the growth of 4T1-Luc tumours compared to the controls. n = 13 biological replicates for saline, n = 14 biological replicates for NE, n = 13 biological replicates for NE + CAMP. Data represented as mean ± SEM. Analysed by ordinary two-way ANOVA with Sidak’s multiple comparisons tests. c, Survival in the 4T1-Luc model. Neutrophil-CAMPs significantly improved the survival with 2/13 mice achieving complete regression by day 40. Analysed by log-rank (Mantle-Cox) test.

Source data

Extended Data Fig. 5 Systemic dose of CAMP-carrying neutrophils reduces tumor burden in the B16F10 model synergistically with aPD-1.

a, Experimental scheme for the treatment of B16F10 tumours. In addition to the two doses of saline/NE/NE+CAMPs, mice were treated with 100 μg intraperitoneal dose of aPD-1 antibody, 4 times, 3 days apart. b, Tumour growth kinetics of various aPD-1 combination treatments in the B16F10 model. Data represented as mean ± SEM. Analysed by ordinary two-way ANOVA with Sidak’s multiple comparisons tests. c, Survival in the B16F10 model for aPD-1 combination treatments. Analysed by log-rank (Mantle-Cox) test. d, Individual tumour growth curves for each group. n = 6 biological replicates for Saline + aPD-1, n = 6 biological replicates for NE + aPD-1, n = 11 biological replicates for NE + CAMP + aPD-1. CR represents complete regression.

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Source Data Fig. 6 and Extended Data Figs. 4 and 5

Source data for tumour progression and for survival for Fig. 6 and Extended Data Figs. 4 and 5.

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Kumbhojkar, N., Prakash, S., Fukuta, T. et al. Neutrophils bearing adhesive polymer micropatches as a drug-free cancer immunotherapy. Nat. Biomed. Eng (2024). https://doi.org/10.1038/s41551-024-01180-z

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