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1 In situ vaccination with cowpea mosaic virus nanoparticles suppresses metastatic cancer P. H. Lizotte, A. M. Wen, M. R. Sheen, J. Fields, P. Rojanasopondist, N. F. Steinmetz, S. Fiering Supplementary Information NATURE NANOTECHNOLOGY 1

2 Supplementary Fig. 1. ecpmv particles inhaled into WT mouse lung induces activation of quiescent neutrophils. a, Neutrophils taking up ecpmv upregulated expression of cell surface CD11b, indicative of activation. ecpmv inhalation also induced infiltration of conventional F4/80+class-II+ macrophages. b-c, Graphs pooling multiple replicates of a indicating conversion of quiescent neutrophils to activated neutrophils and influx of macrophages. 2 NATURE NANOTECHNOLOGY

3 Supplementary Fig. 2. ecpmv particle is taken up by activated neutrophils that express CD11b and CD86 co-stimulatory molecule in the WT mouse lung. Representative histograms of Alexa488-labeled CPMV nanoparticle uptake, MHC class-ii, and CD86 expression. NATURE NANOTECHNOLOGY 3

4 Supplementary Fig. 3. Gating strategies and nomenclature for B16F10 lung tumorbearing mice described in results section. 4 NATURE NANOTECHNOLOGY

5 Supplementary Fig. 4. Inhalation of ecpmv does not cause visible damage by histology of lungs. a, There was no significant lung damage after intratracheal injection of ecpmv or PBS. H&E staining of lung sections of PBS-treated mice (top) or 100 μg ecpmv-treated mice (bottom) 24hr post-intratracheal injection revealed no significant difference between the two groups as assessed by either alevolar wall thickening or immune cell infiltration. b, histology on other major organs of mice receiving intratracheal injection of ecpmv or PBS. Pictures of H&E staining were taken with X100 magnification. Pictures are representative (n=4). Images were acquired and captured by using the Olympus BX5.1 microscope and Image Pro software. NATURE NANOTECHNOLOGY 5

6 Supplementary Fig 5. 4T1-luc primary flank tumors comparable between groups. BALB/c mice had same primary tumor burden on day 16 prior to surgical removal. Differences in lung metastatic burden in ecpmv- and PBS-treated mice was due to treatment, not to differences between initial tumors. 6 NATURE NANOTECHNOLOGY

7 Supplementary Fig. 6. ecpmv treats tumors better than other TLR or STING agonist adjuvants. Mice bearing B16F10 flank tumors were treated by intratumoral injection on days 12, 14, and 16 with indicated compounds and dosages. LPS, dsrna mimic poly(i:c), and STING agonist DMXAA were inferior to ecpmv as they did not elicit durable anti-tumor responses. Experimental treatments for all groups began once tumors had reached 10mm2. Two-way ANOVA was used for statistical analysis. NATURE NANOTECHNOLOGY 7

8 Supplementary Fig. 7. ecpmv particles are not directly tumoricidal. B16F10, 4T1-luc, and CT-26 cell viability and proliferation are not affected by treatment with ecpmv particles in vitro. Cells were treated with 20μg/well ecpmv in 200 μl complete media. 8 NATURE NANOTECHNOLOGY

9 Supplementary Fig 8. Preparation and characterization of ecpmv. a, External (left) and internal (right) view of structure of ecpmv obtained from the Virus Particle Explorer database ( reconstructed using Chimera. b, TEM imaging with 2% uranyl acetate negative staining verified particle intactness. Stain in the inner cavity of ecpmv demonstrated the complete lack of RNA within the particles. c, UV/visible spectroscopy of the sample with inset zoomed in had a protein peak at 280 nm, with no observed peak at 260 nm that would result from contaminating nucleic acids. d, FPLC analysis confirmed particle intactness and lack of contaminants in sample, as seen from a low A260:A280 ratio. e, Non-denaturing agarose gel electrophoresis of native particles further established the lack of nucleic acids in ecpmv. Ethidium bromide gels visualized under UV light (left) revealed presence of nucleic acids in bacteriophage Qβ VLP and WT CPMV (#1), but not in ecpmv (#2). Gels after Coomassie staining of protein capsids (right) shows localization of ecpmv (#2). The broad band for Qβ results from random encapsulation of different sizes of host RNA. The two bands seen with CPMV (#1) and ecpmv are due to two electrophoretic forms for intact particles, slow and fast, with the fast form resulting from loss of 24 amino acids at the C-terminus of the small coat protein by proteolysis in plants. f, SDS gel electrophoresis after Coomassie staining demonstrated that ecpmv has the same coat protein components as wt CPMV. g, Western blotting verified coat protein composition. M = SeeBlue Plus2 molecular weight marker, 1 = CPMV, 2 = ecpmv. NATURE NANOTECHNOLOGY 9

10 Supplementary Fig. 9. Neutrophil depletion validation. Histograms of lung cells from isotype treated mice (left) compared to anti-ly6g depleted (right). C57BL/6 lung cells harvested 24hr post-ip injection. 10 NATURE NANOTECHNOLOGY