Near Infrared Photoimmunotherapy Targeting EGFR Positive Triple Negative Breast Cancer: Optimizing the Conjugate-Light Regimen

Abstract

Aim: Triple-negative breast cancer (TNBC) is considered one of the most aggressive subtypes of breast cancer. Near infrared photoimmunotherapy (NIR-PIT) is a cancer treatment that employs an antibody-photosensitizer conjugate (APC) followed by exposure of NIR light for activating selective cytotoxicity on targeted cancer cells and may have application to TNBC. In order to minimize the dose of APC while maximizing the therapeutic effects, dosing of the APC and NIR light need to be optimized. In this study, we investigate in vitro and in vivo efficacy of cetuximab (cet)-IR700 NIR-PIT on two breast cancer models MDAMB231 (TNBC, EGFR moderate) and MDAMB468 (TNBC, EGFR high) cell lines, and demonstrate a method to optimize the dosing APC and NIR light.

Method: After validating in vitro cell-specific cytotoxicity, NIR-PIT therapeutic effects were investigated in mouse models using cell lines derived from TNBC tumors. Tumor-bearing mice were separated into 4 groups for the following treatments: (1) no treatment (control); (2) 300 μg of cet-IR700 i.v., (APC i.v. only); (3) NIR light exposure only, NIR light was administered at 50 J/cm2 on day 1 and 100 J/cm2 on day 2 (NIR light only); (4) 300 μg of cet-IR700 i.v., NIR light was administered at 50 J/cm2 on day 1 after injection and 100 J/cm2 of light on day 2 after injection (one shot NIR-PIT). To compare different treatment regimens with a fixed dose of APC, we added the following treatments (5) 100 μg of cet-IR700 i.v., NIR light administered at 50 J/cm2 on day 1 and 50 μg of cet-IR700 i.v. immediately after NIR-PIT, then NIR light was administered at 100 J/cm2 on day 2, which were performed two times every week ("two split" NIR-PIT) and (6) 100 μg of cet-IR700 i.v., NIR light was administered at 50 J/cm2 on day 1 and 100 J/cm2 on day 2, which were performed three times per week ("three split" NIR-PIT).

Result: Both specific binding and NIR-PIT effects were greater with MDAMB468 than MDAMB231 cells in vitro. Tumor accumulation of cet-IR700 in MDAMB468 tumors was significantly higher (p < 0.05) than in MDAMB231 tumors in vivo. Tumor growth and survival of MDAMB231 tumor bearing mice was significantly lower in the NIR-PIT treatment group (p < 0.05). In MDAMB468 bearing mice, tumor growth and survival was significantly improved in the NIR-PIT treatment groups in all treatment regimens (one shot NIR-PIT; p < 0.05, "two split" NIR-PIT; p < 0.01, "three split" NIR-PIT; p < 0.001) compared with control groups.

Conclusion: NIR-PIT for TNBC was effective regardless of expression of EGFR, however, greater cell killing was shown with higher EGFR expression tumor in vitro. In all treatment regimens, NIR-PIT suppressed tumor growth, resulting in significantly prolonged survival that further improved by splitting the APC dose and using repeated light exposures.

Fig 1. Confirmation of EGFR expression as a target for PIT in MDAMB231 and MDAMB468 cells, and evaluation of in vitro PIT.

(A) Expression of EGFR in MDAMB231 cells was examined with FACS. (B) Expression of EGFR in MDAMB468 cells was examined with FACS. (C) MDAMB231 and MDAMB468 cells were incubated with cet-IR700 for 6 h and observed by microscopy. Fluorescence intensities of MDAMB468 were higher than MDAMB231. Necrotic cell death was observed upon excitation with 2 J/cm² of NIR light (after 15min). Bar = 20 μm. (D) Membrane damage of MDAMB231 cells induced by PIT was measured with PI staining (dead cell count), which increased in a light dose-dependent manner (n = 5, *p < 0.001, vs. untreated control, by Student’s t test). (E) PI staining showed membrane damage of MDAMB468 cells (n = 5, *p < 0.001, vs. untreated control, by Student’s t test).

Fig 2. In vivo fluorescence imaging of MDAMB231 and MDAMB468 tumors.

(A) In vivo cet-IR700 and mouse IgG-IR800 fluorescence real-time imaging of bilateral (right dorsum; MDAMB468, left dorsum; MDAMB231) flank tumors in mice. The fluorescence intensity of cet-IR700 and mouse IgG-IR800 in both MDAMB231 and MDAMB468 tumor decreased gradually over days. The fluorescence intensity of IR700 was higher in MDAMB468 tumor compared with MDAMB231 tumor, while the fluorescence intensity of IR800 was almost same in both tumors. (B) Quantitative analysis of IR700 intensities in both tumors. The fluorescence intensities were significantly higher in MDAMB468 tumors compared with MDAMB231 tumors (n = 10, *p < 0.05, **p < 0.01, by Mann-Whitney-U test). (C) Quantitative analysis of TBR in both tumors demonstrated differences between MDAMB468 tumors and MDAMB231 tumors (n = 10, *p < 0.05, **p < 0.01, by Mann-Whitney-U test). (D) Quantitative analysis of TBR700/TBR800 in both tumors. Intensities were significantly higher in MDAMB468 tumors compared with MDAMB231 tumors (n = 10, *p < 0.05, **p < 0.01, by Mann-Whitney-U test).

Fig 3. In vivo effect of NIR-PIT for MDAMB231 tumor.

(A) NIR-PIT regimen. Fluorescence images were obtained at each time point as indicated. (B) In vivo fluorescence real-time imaging of tumor bearing mice in response to NIR-PIT. The tumor treated by NIR-PIT showed decreasing IR700 fluorescence after NIR-PIT. (C) Tumor growth was significantly inhibited in the PIT treatment groups with cet-IR700 (n = 10–13, *p < 0.05 vs i.v. group, **p < 0.01 vs control and light only group, Bonferroni’s test with ANOVA). (D) Significantly prolonged survival was observed in the NIR-PIT treatment group with cet-IR700 (n = 10–13, *p < 0.05 vs other groups, by Log-rank test).

Fig 4. In vivo effect of NIR-PIT for MDAMB468 tumor (one shot NIR-PIT).

(A) NIR-PIT regimen. Fluorescence images were obtained at each time point as indicated. (B) In vivo fluorescence real-time imaging of tumor bearing mice in response to NIR-PIT. The tumor treated by NIR-PIT showed decreasing IR700 fluorescence after NIR-PIT. (C) Tumor growth was significantly inhibited in the NIR-PIT treatment groups with cet-IR700 (n = 10–11, **p < 0.01 vs control and light only group, Bonferroni’s test with ANOVA). (D) Significantly prolonged survival was observed in the APC i.v. only group and the NIR-PIT treatment group with cet-IR700 (n = 10–11, *p < 0.05 vs control, ^# p < 0.05 vs i.v. group, **p < 0.001 vs control and light only group, by Log-rank test).

Fig 5. In vivo effect of PIT for MDAMB468 tumor (“two split” NIR-PIT).

(A) NIR-PIT regimen. (B) Tumor growth was significantly inhibited in the NIR-PIT treatment groups (n = 10–13, **p < 0.01 vs other groups, Bonferroni’s test with ANOVA). (C) Significantly prolonged survival was observed in the APC i.v. only group and the NIR-PIT treatment group (n = 10–13, **p < 0.01 vs other groups, by Log-rank test).

Fig 6. In vivo effect of NIR-PIT for MDAMB468 tumor (“three split” NIR-PIT).

(A) NIR-PIT regimen. (B) Tumor growth was significantly inhibited in the NIR-PIT treatment groups (n = 10–13, **p < 0.001 vs other groups, Bonferroni’s test with ANOVA). (C) Significantly prolonged survival was observed in the APC i.v. only group and NIR-PIT treatment group (n = 10–13, **p < 0.001 vs other groups, by Log-rank test).