Quantitative Imaging of Tumor Associated Macrophages and Their Response to Therapy Using 64Cu-Labeled Macrin

Transcription

1 Supporting Information for Quantitative Imaging of Tumor Associated Macrophages and Their Response to Therapy Using 64Cu-Labeled Macrin Hye-Yeong Kim 1,2+, Ran Li 1+, Thomas S.C. Ng 1, Gabriel Courties 1, Christopher Blake Rodell 1, Mark Prytyskach 1, Rainer H. Kohler 1, Mikael J. Pittet 1,2, Matthias Nahrendorf 1,2, Ralph Weissleder 1,2,3 *, Miles A. Miller 1,2 * 1. Center for Systems Biology, Massachusetts General Hospital Research Institute, Boston, MA Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Boston, MA Department of Systems Biology, Harvard Medical School, Boston, MA equal contribution * Correspondence: miles.miller@mgh.harvard.edu ralph_weissleder@hms.harvard.edu

2 Fig. S1. Optimized Macrin chemistry and functionalization. a) Macrin synthetic scheme. b- c) Macrin diameter (b) and PDI (c) were monitored at four different temperatures during the 5 h cross-linking reaction. d-g) Macrin diameter (d), PDI (e), zeta-potential (f), and amination (g) were measured as a function of cross-linking reaction time. Data are means ± s.e.m. (n > 2 replicates). Note minimal difference in Macrin properties between 5 h and 18 h of reaction time.

3 Fig. S2. Purity, optical and radiochemical properties, and stability of Macrin labeling. a) Radiochemical purity of Macrin in thin layer chromatograms. b-c) Stability of Macrin in mouse serum was measured after 24 h incubation at 37 o C, detected by TLC (b) and SEC (c). d) SEC of VT680XL-Macrin product and VT680XL-NHS reactant. e) Absorption and emission spectra of VT680XL-labeled Macrin in water. f) Macrin, NODA-GA-Macrin, VT680-Macrin, and Cu- Macrin were characterized by DLS. g) DLS, SEM (scale bar, 20 nm), and material characterization of NODA-GA-Macrin. h) NODA-GA-Macrin in buffer (PBS) and in a lyophilized form were monitored at room temperature and at 4 ºC (blue) by DLS. Sizes consistently fall within 10 nm range in hydrodynamic diameter (green shading).

4 Fig. S3. a) Blood half life of 64 Cu-Macrin in wild type (WT) C57BL/6 mice (n = 3). b) Correlation between radioactive decay half-life and optimized circulation half-life (C57BL/6 mice) for various dextran nanoformulations and their respective radionuclides. c) 64 Cu-Macrin biodistribution in MC38 tumor xenografts at 24 h post-injection. Values at right (blue) denote relative Macrin accumulation in Mertk GFP/+ reporter mice compared to age- and sex-matched C57BL/6 for the indicated organs (P > 0.05 in all cases; data are means ± s.d., n = 3, two-tailed t-test). d) Representative prone and supine views of Macrin PET/CT in MC-38 tumor bearing mice (see Fig. 1b). e) Macrin uptake was averaged across all analyzed organs of C57BL/6 and Mertk GFP/+ mice, 24 h post-injection, to assess differences in systemic accumulation (see S3c; student s two-tailed t-test, n=3).

5 Fig. S4. Detailed imaging of Macrin uptake by macrophages. a-b) High magnification in vivo confocal microscopy (intravital) images of VT680XL-labeled Macrin in MC38 tumor bearing MertkGFP/+ reporter mice, 24 h post-injection (scale bar, 10 µm; corresponding to Fig. 1c). c-d) RAW264.7 macrophages were cultured in vitro and treated with various concentrations of Macrin. Cellular uptake was assessed by fluorescence microscopy. c) Representative images of macrophages (scale bar = 100 µm) treated with 1 nmol, 10 nmol (imaging dose, 740 µg of Macrin, orange), and 100 nmol of VT680-Macrin. d) Corresponding quantification of Macrin uptake showing dose dependent uptake (n=50 cells per group; data are means ± s.e.m.).

6 Fig. S5. Flow cytometry analysis of Macrin uptake in macrophages across tissues. Seven organs (bone marrow, spleen, heart, kidney, fat, lung, and liver) and blood were collected at 24 h after i.v. administration of Macrin to MC38 tumor-bearing mice (n=3). From CD45+ cells, macrophages were identified as a combination of CD11b high/int, F4/80 high, and in the lung, CD64+, as indicated by the gating schemes shown. The mean fluorescent intensity (MFI) of Macrin in macrophages from tumor-bearing mice (blue) was quantified in histograms and compared to the vehicle control in the matched cell population (grey). Circulating leukocytes were also identified, showing no macrophages or Macrin cellular accumulation. Both Kupffer and perivascular macrophages were detected in the liver, along with interstitial and alveolar macrophages in the lung. Fig. 3 presents data for Kupffer cells and interstitial macrophages.

7 Fig. S6. Macrin localization in tissue macrophages at 24 h post-injection. Representative confocal fluorescence images and quantitative analysis of Macrin in liver (a) and in lymph nodes (b) from Mertk GFP/+ reporter mice. Scale bars 10 µm (left; high magnification) and 50 µm (low magnification), respectively, showing relative accumulation in GFP + vs GFP - cell populations.

8 Fig. S7. Heterogeneous Macrin accumulation across KP lung tumors. a) Confocal microscopy images of the lung displayed in Fig. 4e (inset scatter plot), annotated with region of the interests (ROIs) used to quantify Macrin signals. b) Macrin uptake on a per-cell basis was quantified as a function of tumor size using the orthotopic KP model (C57BL/6 mice), 24 h post Macrin injection, imaged with confocal microscopy after organ harvesting and optically clearing (data are means ± s.e.m. for single cells within n = 6 tumors, Spearman correlation R and P values reported). c) At the macroscopic level (e.g., 5d), 27 individual tumor regions in KP tumorbearing Cx3cr1-GFP reporter mice (C57BL/6 background) were quantified for both Macrin and GFP (data are from n~25 tumor regions, Spearman correlation R and P values reported). Note the lack of {GFP high, Macrin low} regions.

9 Fig. S8. Analysis of TAM heterogeneity following neo-adjuvant treatment in KP1.9 lung adenocarcinoma models. KP tumor-bearing lungs from C57BL/6 mice were optically cleared and imaged with confocal microscopy to measure fluorescent VT680-labeled Macrin uptake within each tumor nodule. a-b). Single-dose RT and OC treatments did not significantly change tumor sizes (a), but enhanced TAM levels in the tumors of similar sizes (b). Data are from 733 tumors across 14 lungs. c-d) Radial intensity profiles of VT680-labeled Macrin accumulation were measured as a function of distance to the tumor edge (c; thick line and shading are means +/- s.e.m., normalized to the maximum average intensity observed across all distances). Relative levels in the tumor interior and at the edge were quantified and observed to increase with RT (e; means +/- s.e.m., ANOVA).

10 Fig. S9. The ability of macrin to predict polymeric nanoparticle uptake is not dependent on tumor size in KP1.9 lung adenocarcinoma. Tumoral uptake of Macrin and PLGA-PEG NP across multiple KP lung tumor nodules (from C57BL/6 mice) of varying sizes was examined. Each tumor nodule was categorized as small (diameter, d<0.25 mm, red), medium (0.25 mm<d<0.35 mm, blue), or large (d>0.35 mm, green). Similar correlations between Macrin and PLGA-PEG NP uptake were observed for all three sub-categories.

11 Fig. S10. Macrin preferentially targets macrophages over tumor cells. MC38 tumor cells or bone-narrowed derived macrophages (BMDM, MΦ) were treated with VT680-Macrin, and the accumulation of Macrin in these cells were compared with fluorescent microscopy. a) Representative fluorescence microscopy showing Macrin accumulate within the macrophages (either untreated or treated with various cytokines) compared to MC38 tumor cells (scale bar=100 µm). b) Quantification of images in a, showing macrophages uptake, on average, 20 times more Macrin than MC38 tumor cells.