Low dose gemcitabine-loaded lipid nanocapsules target monocytic myeloid-derived suppressor cells and potentiate cancer immunotherapy

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Supplementary Figures

Supplementary Fig. S1. Biodistribution of DiD-loaded LNCs. Mice bearing ~50mm² EG7-OVA tumors were injected with DiD-loaded LNCs either IV (on the tail vein) or SC (on the flank opposite to the tumor injection site). A. Average DiD fluorescence signal in explanted organs at sequential time points following LNC injection. Means ± SD; n=5 mice per group. B, C, D, E. Representative confocal microscopy images of tissue sections at 24 hours from DiD-LNC injection.

Lymphoid follicles in the spleen are identified as zones with high nuclei density surrounded by CD11b⁺ myeloid cells.

Nuclei in gray (DAPI staining), CD11b in green, DiD in blue. 40x magnification. B. Spleen after DiD-LNC IV injection. C.

Spleen after DiD-LNC SC injection. D. Tumor after DiD-LNC IV injection. E. Tumor after DiD-LNC SC injection. F. Mean

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Fluorescence intensity (MFI) of DiD in spleens and tumor sections, calculated on a set of 4-7 randomly selected fields per section. Means± SE, n= 3 mice per group. *p<0.05 **p<0.01, Student’s t-test.

Supplementary Fig. S2. Gating strategy for the identification of myeloid cell populations in the spleen and tumor. A. Dot plots showing the gating of red pulp macrophages (CD11b^low/negF4/80^high), polymorphonuclear/granulocytic (PMN-) MDSCs (CD11b⁺Ly6G⁺Ly6C^int) and monocytic (M-) MDSCs (CD11b⁺Ly6G^-Ly6C^high) in the spleen. M-MDSCs were also F4/80^low, and partially expressed the CD115 marker. B. Gating of DCs in the spleen (CD11c⁺MHCII⁺). C. Gating of PMN- MDSCs (CD11b⁺Ly6G⁺Ly6C^int), M-MDSCs (CD11b⁺Ly6G^-Ly6C^high) and macrophages (CD11b⁺Ly6G^-Ly6C^low/-F4/80⁺) in the tumor. All analyses were performed after selection of living single cells by morphologic gating, doublets exclusion and dead cell exclusion by LIVE/DEAD® dye staining. Reported dot plots refer to spleens and tumors of C57BL/6 mice

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Supplementary Fig. S3. GemC12-LNC treatment does not affect liver macrophages. Representative dot plots (A) and bar charts (B) reporting the frequency of F4/80⁺CD11b^low and F4/80⁺CD11b^high liver macrophages (Kupffer cells) 24 hours following the SC administration of indicated treatments. Means± SE, n= 5 mice per group. Kupffer cell subsets were gated as CD45⁺, Ly6G^- F480⁺ CD11b^lowand CD45⁺, Ly6G^- F480⁺ CD11b^high, as reported in [1-3].

Supplementary Fig. S4. Frequency of MDSCs at one week from GemC12-LNC treatment. Frequency of M-MDSCs, PMN-MDSCs ad macrophages in the tumor (A) and spleen (B) one week following low dose GemC12-LNC SC injection.

Means± SE, n= 3 mice per group.

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Supplementary Fig. S5. Treatment with low dose GemC12-LNCs or GemHCl, without ACT, does not improve mouse survival. Drug treatments were administered at a dose of 11 mg/kg GemHCl (or molar equivalent GemC12-LNCs) to mice bearing EG7-OVA tumors, at day 8 following tumor cell injection. N=5 mice per group. Survival curves were not significantly different (p>0.05, Log-Rank test).

Supplementary Methods

Tracking of DiD-loaded-LNCs in tumor-bearing mice

Mice bearing ~50 mm

²

EG7-OVA tumors were injected with DiD-loaded LNCs either subcutaneously, on the

right flank, or intravenously, into the tail vein. For both biodistribution studies and cell uptake studies, mice

were injected with 12,7 mg of DiD-LNCs (~6 μg of DiD dye), corresponding to the same amount of LNC per

mouse administered in experiments with GemC12-loaded LNCs. For biodistribution experiments, mice were

sacrificed at sequential time points from DiD-LNC injection (24h, 48h, 72h) and organs (i.e. kidneys, liver,

spleen, lung, heart, stomach, intestine and tumor) were removed and analyzed by a fluorescence CRI

Maestro TM imaging system (Woburn, USA). Semi-quantitative data were obtained by setting a time

exposition of 10 msec between 630 and 800 nm, unmixing the generated cube, extracting the background

and drawing the regions of interest from fluorescence images. The software Maestro 2.10 (Woburn, USA)

was used to calculate the average signal expressed in photon/cm²/s.

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were re-hydrated in PBS, fixed in 4% PFA for 5 minutes at room temperature (RT), incubated 1h RT with a blocking solution (PBS 10% FBS) to reduce non-specific binding, and stained overnight with rat anti-mouse CD11b-FITC (clone M1/70 eBioscience). Goat anti-rat IgG Alexa Fluor 488 (Abcam) was used as secondary antibody and incubated 3 hours at RT. Nuclear staining was performed with DAPI (Invitrogen) for 10 minutes at RT. Slices were mounted with ProLongR Gold Antifade Reagent (Invitrogen). Images were acquired with a Zeiss LSM 510 confocal microscope. MFI in the DiD channel was measured on 4-7 randomly selected fields per tissue section, and average MFI ± standard errors (SE) was calculated for tissue sections from n=3 mice per group.

Supplementary References

[1] Kinoshita M, Uchida T, Sato A, Nakashima M, Nakashima H, Shono S, et al. Characterization of two F4/80- positive Kupffer cell subsets by their function and phenotype in mice. Journal of hepatology. 2010;53:903- 10.

[2] Scott CL, Zheng F, De Baetselier P, Martens L, Saeys Y, De Prijck S, et al. Bone marrow-derived monocytes give rise to self-renewing and fully differentiated Kupffer cells. Nature communications. 2016;7:10321.