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A lysosome-targeted DNA nanodevice selectively targets macrophages to attenuate tumours

Nature Nanotechnology volume 16, pages 1394–1402 (2021)Cite this article

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Abstract

Activating CD8+ T cells by antigen cross-presentation is remarkably effective at eliminating tumours. Although this function is traditionally attributed to dendritic cells, tumour-associated macrophages (TAMs) can also cross-present antigens. TAMs are the most abundant tumour-infiltrating leukocyte. Yet, TAMs have not been leveraged to activate CD8+ T cells because mechanisms that modulate their ability to cross-present antigens are incompletely understood. Here we show that TAMs harbour hyperactive cysteine protease activity in their lysosomes, which impedes antigen cross-presentation, thereby preventing CD8+ T cell activation. We developed a DNA nanodevice (E64-DNA) that targets the lysosomes of TAMs in mice. E64-DNA inhibits the population of cysteine proteases that is present specifically inside the lysosomes of TAMs, improves their ability to cross-present antigens and attenuates tumour growth via CD8+ T cells. When combined with cyclophosphamide, E64-DNA showed sustained tumour regression in a triple-negative-breast-cancer model. Our studies demonstrate that DNA nanodevices can be targeted with organelle-level precision to reprogram macrophages and achieve immunomodulation in vivo.

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Fig. 1: M2 macrophages have elevated lysosomal enzyme levels and activity.
Fig. 2: Deleting Tfeb in myeloid cells attenuates tumour growth through CD8+ T cell activation.
Fig. 3: Lysosomal cysteine proteases are elevated in M2 macrophages.
Fig. 4: A lysosome-targeted DNA nanodevice (E64-DNA) promotes antigen cross-presentation by TAMs.
Fig. 5: The E64-DNA nanodevice preferentially localizes in lysosomes of M2-like TAMs and lowers tumour growth.
Fig. 6: Intravenously delivered E64-DNA targets TAMs to activate CD8+ T cells and attenuate tumour growth.

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Data availability

Source data are provided with this paper. All data generated or analysed during this study are included in this published article and its Supplementary information files. Proteomics data are available via ProteomeXchange with identifier PXD028037.

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Acknowledgements

We thank N. Hansen, K. Bethke and S. Khan, as well as B. LaBomascus at Northwestern University for assistance with obtaining tumours from ER+ breast cancer patients. Tfebfl/fl mice were a gift from A. Ballabio, Telethon Institute of Genetics and Medicine. pMel and TRP1 mice were a gift from M. Swartz, University of Chicago. E0771 cells were a gift from M. Rosner, University of Chicago. B16F10 cells were a gift from T. Gajewski, University of Chicago. B16.OVA cells were a gift from J. Hubbell, University of Chicago. This work was supported by the Women’s Board Faculty Research Startup Funds and Ben May Department Startup Funds (L.B.), the University of Chicago Women’s Board (Y.K.) and the Ono Pharma Breakthrough Science Award (Y.K.). C.C. was supported by a Bernice Goldblatt Scholarship. K.C. was supported by a Schmidt Science Fellowship, in partnership with the Rhodes Trust. B.M. was supported by the National Cancer Institute (R25CA221767).

Author information

Authors and Affiliations

  1. Committee on Cancer Biology, The University of Chicago, Chicago, IL, USA

    Chang Cui & Lev Becker

  2. Ben May Department for Cancer Research, The University of Chicago, Chicago, IL, USA

    Chang Cui, Kasturi Chakraborty, Xu Anna Tang, Kelly Q. Schoenfelt, Alexandria Hoffman, Ariane Blank, Blake McBeth, Catherine A. Reardon & Lev Becker

  3. Department of Chemistry, The University of Chicago, Chicago, IL, USA

    Kasturi Chakraborty & Yamuna Krishnan

  4. Department of Surgery, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    Natalie Pulliam & Swati A. Kulkarni

  5. UW Medicine Diabetes Institute, University of Washington, Seattle, WA, USA

    Tomas Vaisar

  6. Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy

    Andrea Ballabio

  7. Department of Translational Medicine, Federico II University, Naples, Italy

    Andrea Ballabio

  8. Neurological Research Institute, Baylor College of Medicine, Houston, TX, USA

    Andrea Ballabio

  9. Grossman Institute of Neuroscience, Quantitative Biology and Human Behavior, The University of Chicago, Chicago, IL, USA

    Yamuna Krishnan

  10. Ludwig Center for Metastasis Research, The University of Chicago, Chicago, IL, USA

    Lev Becker

  11. University of Chicago Comprehensive Cancer Center, The University of Chicago, Chicago, IL, USA

    Lev Becker

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  1. Chang Cui
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Contributions

C.C., K.C., Y.K. and L.B. conceived and designed the experiments. C.C., K.C., X.A.T., K.Q.S., A.H., A. Blank, B.M., C.A.R. and T.V. performed the experiments: A. Ballabio provided material support. N.P. and S.A.K. provided the patient tumours. All authors critically reviewed the manuscript. C.C., Y.K. and L.B. wrote the manuscript.

Corresponding authors

Correspondence to Yamuna Krishnan or Lev Becker.

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Competing interests

L.B., Y.K., C.C. and K.C. are inventors on a provisional patent (related to DNA-based therapeutics delivery) filed by the University of Chicago. L.B. and Y.K. are co-founders of MacroLogic, a startup biotechnology company focused on developing DNA-conjugated therapeutics. A. Ballabio is a co-founder of CASMA Therapeutics and is an Advisory Board member of Next Generation Diagnostics and of Avilar Therapeutics. X.A.T., K.Q.S., A.H., A. Blank, B.M., N.P., C.A.R., S.A.K. and T.V. declare no competing interests.

Additional information

Peer review information Nature Nanotechnology thanks Chunhai Fan, Lisa Sevenich and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 TFEB is responsible for elevated lysosomal enzymes in M2-like macrophages.

a, Validation of lysosomal proteins elevated in M2 BMDMs by immunoblotting, related to Fig. 1d. Representative of 2 independent experiments. b, mRNA levels of lysosomal genes in M1 and M2 BMDMs. n = 3/group. c, Tfeb mRNA levels in M1 and M2 BMDMs. n = 3/group. d, Immunoblot of TFEB protein levels in M1 and M2 BMDMs. Representative of 3 independent experiments. e, Immunoblot of cytosolic and nuclear TFEB levels in M1 and M2 BMDMs. Representative of 2 independent experiments. f, Validation of mTfeb-/-. mRNA levels (top) n = 3/group and protein levels (bottom). Representative of 3 independent experiments. g, A comparison of lysosomal gene expression in M1 and M2 BMDMs from fl/fl mice versus M2 BMDMs from mTfeb-/- mice, n = 3/group; and a comparison of lysosomal gene expression in TAMs from fl/fl and mTfeb-/- E0771 tumors, n = 4/group. h, DQ-OVA degradation assays of fl/fl and mTfeb-/- M2 BMDMs. n = 3/group. Statistical significance was calculated via two-tailed Student’s t-test (P < 0.05 values are provided); error bars indicate the mean of independent experiments ± s.e.m. All measurements (n) are biological replicates.

Source data

Extended Data Fig. 2 TAMs exhibit increased lysosomal enzyme levels and activity.

a, Isolation of mammary ATMs from tumor-free mice and TAMs from E0771 mammary tumor-bearing mice. Purity of ATMs and TAMs was validated by flow cytometry. b, Immunoblots of lysosomal protein levels in ATMs and TAMs. Experiment was performed once with n = 3/group. c, DQ-OVA degradation assays of ATMs and TAMs. n = 3/group. d, mRNA expression of lysosomal genes in TAMs isolated from E0771 tumors and thioglycolate-elicited peritoneal macrophages from tumor-free mice. n = 3/group. Statistical significance was calculated via two-tailed Student’s t-test (P < 0.05 values are provided); error bars indicate the mean of independent experiments ± s.e.m. All measurements (n) are biological replicates.

Source data

Extended Data Fig. 3 TAMs from mTfeb-/- mice exhibit improved antigen cross-presentation with minimal phenotypic changes.

a-c, TAMs were isolated from E0771 tumors. a, Quantification of lysosomes in fl/fl and mTfeb-/- TAMs based on LAMP1 immunostaining. Schematic for quantification (left). Quantification of average LAMP1 signal/cell area (n = 10/group) with an average of >40 cells/field (middle). Representative images (right). LAMP1 (red) and DAPI (blue). b, Quantification of lysosomal pH in fl/fl and mTfeb-/- TAMs based on lysotracker staining. Representative flow cytometry image (left). Quantification of relative MFI of lysotracker signal (right). n = 3/group. c, Autophagy gene expression in fl/fl and mTfeb-/- TAMs (left, n = 5). LC3B and p62 protein levels in fl/fl and mTfeb-/- TAMs following treatment with vehicle (Veh) or chloroquine (CQ, 50μM) for 24 h (right). Veh = H2O. Experiment was performed once with n = 3/group. d, M1- and M2-associated gene expression in TAMs from fl/fl and mTfeb-/- E0771 tumors (left, n = 5/group), LLC1 tumors (middle, n = 5/group) and B16F10 tumors (right, n = 4 group). e-f, Quantification of pMel-CD8+ T cell activation (e) and proliferation (f) following co-culture with TAMs isolated from fl/fl and mTfeb-/- B16.OVA tumors. n = 6/group Statistical significance was calculated via two-tailed Student’s t-test (P < 0.05 values are provided); error bars indicate the mean of independent experiments ± s.e.m. ns; not significant. All measurements (n) are biological replicates.

Source data

Extended Data Fig. 4 Deleting Tfeb in myeloid cells attenuates tumor growth via CD8+ T cells (B16F10 & LLC1 models).

a, B16F10 tumor growth rates in fl/fl (n = 14) and mTfeb-/- (n = 10) mice (left). LLC1 tumor growth rates in fl/fl (n = 10) and mTfeb-/- (n = 8) mice (right). b, Tumor immune cell composition in B16F10 tumor bearing fl/fl (n = 8) and mTfeb-/- (n = 6) mice; Tumor immune cell composition in LLC1 tumor bearing fl/fl (n = 9) and mTfeb-/- (n = 8) mice. CD8+ Teff = effector CD8+ T cells. c, Blood CD8+ T cell levels in mice treated with α-CD8 or IgG antibodies. Representative flow cytometry data (left). Quantification of CD8+ and CD4+ T cells (right). n = 4/group. d, Final tumor volume in B16F10 (n = 5/group) and LLC1 (fl/fl: n = 6, mTfeb-/-: n = 7 (IgG), n = 6 (α-CD8)) tumor bearing fl/fl and mTfeb-/- mice treated with IgG or α-CD8 antibodies. Statistical significance was calculated via two-tailed Student’s t-test (P < 0.05 values are provided); error bars indicate the mean of independent experiments ± s.e.m. ns; not significant. All measurements (n) are biological replicates.

Extended Data Fig. 5 DNA nanodevice uptake and stability.

a, Schematic of various fluorescently labeled nucleic acid structures used for uptake studies in BMDMs. Each nucleic acid scaffold is either a single stranded or double stranded 38 mer DNA or RNA sequence. Each scaffold is labelled with an Alexa Fluor® 647 fluorophore on the 5′ end of one of the strands. b, Uptake of various types of nucleic acids by M2 BMDMs. n = 3/group. c, Native polyacrylamide gel of dsDNA incubated in 100% mouse serum for various time points. Intact dsDNA was quantified by densitometry. Representative of 2 independent experiments. d, Schematic of an E64 − DNA uptake competition assay in M1 and M2 BMDMs. e, Hoechst dye levels in individually cultured M1 and M2 BMDMs. f, E64 − DNA uptake by co-cultured M1 and M2 BMDMs. Representative flow cytometry data (left) and quantification (right) are shown. n = 3/group. Statistical significance was calculated via two-tailed Student’s t-test (P < 0.05 values are provided); error bars indicate the mean of independent experiments ± s.e.m. ns; not significant. All measurements (n) are biological replicates.

Source data

Extended Data Fig. 6 Effects of E64-DNA on the functional properties of TAMs.

a, Catalytic activity assays for lysosomal cysteine proteases (CTSB, CTSL; 5 nM) or aspartic proteases (CTSD, CTSE; 5 nM) in the presence of vehicle (Veh; PBS) or E64-DNA (25 nM). Results are plotted as fluorescence intensity at time t, relative to time 0 (I/Io). n = 3/group. b-d, TAMs isolated from E0771 tumors were treated with vehicle (Veh; PBS), DNA, E64, or E64-DNA (100 nM). b, Cell viability (Calcein-AM) following a 72 h exposure. n = 4/group. c, CTSB and CTSL protein levels following a 24 h exposure. Experiment was performed once with n = 3/group. d, Relative mRNA levels of autophagy genes following a 24 h exposure. n = 3/group. e, LC3B and p62 protein levels in DNA or E64-DNA (10uM) treated TAMs following a 24 h treatment with vehicle (Veh; H2O) or chloroquine (CQ, 50μM). Representative of 2 independent experiments. f, Effect of E64-DNA (2 h) on TBK and IRF3 phosphorylation. TAMs treated with 3’3′-cGAMP (10μg/mL, 6 h) were used as a positive control for STING activation Representative of 2 independent experiments. g, Effect of E64-DNA (24 h) on M1- and M2-associated gene expression. n = 3/group. Statistical significance was calculated via two-tailed Student’s t-test (P < 0.05 values are provided); error bars indicate the mean of independent experiments ± s.e.m. ns; not significant. All measurements (n) are biological replicates.

Source data

Extended Data Fig. 7 E64-DNA does not activate T cells through allostimulation or direct stimulation.

a-b, Control for allostimulation. CD8+ T cell activation (a) and proliferation (b) after 72 h of co-culture with E64-DNA-treated (100 nM) TAMs that had not been exposed to antigen. CD3/CD28 antibodies were included as a positive control for T cell activation. n = 3/group. c-d, Control for direct effects of E64-DNA on T cells. CD8+ T cell activation (c) and proliferation (d) after 72 h of culturing in complete growth media (Media) in the presence/absence of E64-DNA (100 nM). CD3/CD28 antibodies were included as a positive control for T cell activation. n = 3/group. Statistical significance was calculated via two-tailed Student’s t-test (P < 0.05 values are provided); error bars indicate the mean of independent experiments ± s.e.m. All measurements (n) are biological replicates.

Extended Data Fig. 8 Inhibiting aspartic protease activity in the lysosome has minimal effect on antigen cross-presentation by macrophages.

a, PepA-DNA design: one strand is conjugated with PepA on its 5’ end and the other with Alexa Fluor 647 to monitor uptake. b, Catalytic activity assays for lysosomal cysteine proteases (CTSB, CTSL; 5 nM) or aspartic proteases (CTSD, CTSE; 5 nM) in the presence of vehicle (Veh; PBS) or PepA-DNA (25 nM). Results are plotted as fluorescence intensity at time t, relative to time 0 (I/Io). n = 3/group. c–f, Peritoneal macrophages were isolated and treated with vehicle (Veh; PBS), DNA, PepA, or PepA-DNA (100 nM) for the indicated times and various functional endpoints were measured. c, Effect of PepA-DNA (2 h) on DQ-OVA degradation. n = 3/group. d, Quantification of MHCI-bound OVA257-264 on peritoneal macrophages 3 h post treatment with OVA protein or OVA257-264 peptide. n = 3/group. e–f, pMel-CD8+ T cell activation (e) and proliferation (f) after 72 h of co-culture with peritoneal macrophages pre-stimulated with irradiated B16F10 cells (irrB16). n = 3/group. Statistical significance was calculated via two-tailed Student’s t-test (P < 0.05 values are provided); error bars indicate the mean of independent experiments ± s.e.m. ns; not significant. All measurements (n) are biological replicates.

Extended Data Fig. 9 E64-DNA does not improve MHCII-restricted antigen presentation.

Effect of E64-DNA on MHCII-restricted antigen presentation by TAMs (isolated from E0771 tumors) pre-treated with E64-DNA, DNA, or E64 (100 nM) for 2 h. a–d, TAMs were incubated with OVA protein or OVA332-339 peptide for 3 h. OT-2 CD4+ T-cell activation (a–b) and proliferation (c–d) after 72 h of co-culture with TAMs. n = 3/group. e-f, TAMs were incubated with irradiated B16F10 cells (irrB16) or TRP1113-126 peptide for 3 h. TRP1 CD4+ T-cell activation (e) and proliferation (f) after 72 h of co-culture with TAMs. n = 3/group. Statistical significance was calculated via two-tailed Student’s t-test (P < 0.05 values are provided); error bars indicate the mean of independent experiments ± s.e.m. All measurements (n) are biological replicates.

Extended Data Fig. 10 E64-DNA attenuates tumor growth and improves antigen cross-presentation by TAMs in the B16.OVA model.

a, Experimental design (left). Effect of E64-DNA (25μg, i.v.) on B16.OVA tumor growth (right). n = 8/group. b, OT-1-CD8+ T cell activation (left) and proliferation (right) after 72 h of co-culture with TAM isolated from DNA or E64-DNA (i.v.) treated B16.OVA tumors. n = 6/group. c, pMel-CD8+ T cell activation (left) and proliferation (right) after 72 h of co-culture with TAMs isolated from DNA or E64-DNA (i.v.) treated B16.OVA tumors. n = 6/group. d-e. Effects of E64-DNA on CD8+ T cell activation and proliferation status 5 days after a single injection. Representative flow images (d) and quantification (e). n = 9/group. Statistical significance was calculated via two-tailed Student’s t-test (P < 0.05 values are provided); error bars indicate the mean of independent experiments ± s.e.m. All measurements (n) are biological replicates.

Supplementary information

Supplementary Information

Supplementary Figs. 1–4.

Reporting Summary

Supplementary Table 1

Shotgun proteomics analysis of M1 and M2 BMDMs.

Supplementary Table 2

Shotgun proteomics analysis of M1-like and M2-like TAMs from E0771 tumours.

Supplementary Table 3

Primers for PCR analysis.

Supplementary Table 4

DNA nanodevice sequences.

Source data

Source Data Fig. 3

Unprocessed western blots.

Source Data Fig. 4

Unprocessed gels.

Source Data Extended Data Fig. 1

Unprocessed western blots.

Source Data Extended Data Fig. 2

Unprocessed western blots.

Source Data Extended Data Fig. 3

Unprocessed western blots.

Source Data Extended Data Fig. 5

Unprocessed gels.

Source Data Extended Data Fig. 6

Unprocessed western blots.

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Cui, C., Chakraborty, K., Tang, X.A. et al. A lysosome-targeted DNA nanodevice selectively targets macrophages to attenuate tumours. Nat. Nanotechnol. 16, 1394–1402 (2021). https://doi.org/10.1038/s41565-021-00988-z

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