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php12018-sup-0001-FigureS1.pdfapplication/PDF245KFigure S1. In vitro phototoxicity experiments. (a) Phototoxicity experiments with A-431/G4 cells showing that PPa is highly phototoxic in vitro, whereas Ac-sPPp is relatively poorly phototoxic in vitro. Note that increasing the incubation period from 16 to 40 h did not increase the phototoxicity of Ac-sPPp. (b) Phototoxicity experiments showing that at a moderate PDT dose of 150 nm PPa equivalents and 10 J cm−2 of 670 nm light, anti-EGFR Ac-sPPp-cetuximab PIC is highly phototoxic toward EGFR-overexpressing A-431/G4 cells, while the control anti-HER2 Ac-sPPp-trastuzumab PIC exhibits no phototoxicity. In addition, competition of Ac-sPPp-cetuximab PIC with a four-fold greater concentration of native cetuximab largely inhibits all cell killing, demonstrating that Ac-sPPp-cetuximab PIC phototoxicity is mediated through specific binding of the EGFR under the specified moderate PDT treatment conditions. (c) Phototoxicity experiments showing that anti-EGFR Ac-sPPp-cetuximab PIC is only mildly to moderately phototoxic to non-target low EGFR-expressing SK-BR-3 cells, whereas PPa is highly phototoxic. Ac-sPPp-cetuximab phototoxicity towards SK-BR-3 cells is not fully inhibited by competition with native cetuximab, indicating that these cells also take up PIC by nonspecific processes such as adsorptive pinocytosis and/or phagocytosis. (d) Phototoxicity experiments with A-431/G4 cells showing that PIC targeting becomes nonspecific at relatively high PDT doses. For these experiments, cells were incubated with Ac-sPPp or anti-erbB Ac-sPPp PIC for 16 h. The light dose was 20 J cm−2 unless indicated otherwise. Note that at 150 nm PPa equivalents and 20 J cm−2 light dose, PIC targeting still exhibits a high degree of specificity as Ac-sPPp-cetuximab PIC phototoxicity is markedly inhibited by competition with the corresponding native cetuximab but not by competition with negative control trastuzumab. However, at relatively high PDT doses (≥300 nm PPa equivalents and ≥20 J cm−2 light dose), targeting specificity is lost, and PIC phototoxicity is not appreciably inhibited by competing native antibody. Error bars indicate standard deviations and are shown for data that was collected at least in triplicate.
php12018-sup-0002-FigureS1.pdfapplication/PDF4639KFigure S2. Fluorescence time-course imaging with Ac-sPPp-cetuximab PIC. A mouse bearing an A-431 tumor xenograft implanted subcutaneously in the left pectoral region was injected via the tail vein with Ac-sPPp-cetuximab PIC in PBS. Imaging experimental conditions were the same as those described in Fig. 2 where corresponding time-course imaging data for Ac-sPPp and the parent PS, PPa, are shown for comparison.
php12018-sup-0003-FigureS3.pdfapplication/PDF4397KFigure S3. Repeat PDT treatment of A-431 tumor-bearing mice using Ac-sPPp or Ac-sPPp-cetuximab PIC with light doses administered 40 h post-PS injection. Treatment conditions were similar to those described in Fig. 3 except the PS dose given for each individual PDT session was 5 nmol PPa equivalents per g body weight (instead of 3 nmol), and mice were PDT irradiated 40 h post-PS injection (instead of 16 h). (a) Tumor growth response for mice treated with repeat Ac-sPPp PDT (open triangles) or repeat Ac-sPPp-cetuximab PDT (filled triangles). Initial PDT was given day zero and then repeated 6 days later for Ac-sPPp PDT or 8 days later for Ac-sPPp-cetuximab PDT (arrows indicate the timing of the second treatment). Number of mice for each treatment regimen is indicated by n. For comparison, tumor growth curves for a group of untreated control mice (no PS and no light, gray open circles; = 10) are shown along with an average exponential fit of these data (thick line). (b) Representative photographs depicting tumor and surrounding normal tissue response of a mouse over the course of treatment with repeat Ac-sPPp-cetuximab PDT. One of the tumor growth response curves shown in (a) corresponds to this mouse. (c) Fluorescence imaging before and after the first round of Ac-sPPp-cetuximab PDT for the same mouse shown in (b). Photobleaching post-PDT is most prominent at the irradiated tumor site but is also evident elsewhere throughout the animal.

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