Development of an ErbB‐overexpressing A‐431 Optical Reporting Tumor Xenograft Model to Assess Targeted Photodynamic Therapy Regimens

Abstract

To better assess the efficacy of erbB-targeted therapies, it would help to have optical reporting human tumor xenograft models that abundantly express erbB receptors. A-431 cells have frequently been used in erbB1-targeting studies, but a well-characterized optical reporting version of the cell line has not been readily available. In this study, optical reporting A-431 clones were developed that express both a fluorescent protein reporter (green, GFP; or red, RFP) and a bioluminescent reporter, firefly luciferase. Reporter genes were transduced into cells using commercial lentiviral vectors, and clonal selection was carried out using a series of procedures. A number of clones were isolated for further characterization. A GFP/luciferase clone, A-431/D4, and an RFP/luciferase clone, A-431/G4, were obtained that exhibit erbB1 expression levels and tumor growth kinetics similar to the parental cells. To demonstrate the utility of the optical reporting clones, A-431/G4 tumors were grown subcutaneously in nude mice and treated with vascular-targeted photodynamic therapy (PDT), which targets the angiogenic consequences of erbB signaling. The A-431/G4 tumor model permitted highly sensitive longitudinal monitoring of PDT treatment response using optical imaging. A-431/D4 and A-431/G4 optical reporting tumor models should also prove useful for assessing therapies that directly target the erbB1 receptor.

Photodynamic Tumor Eradication With a Novel Targetable Photosensitizer: Strong Vascular Effects and Dependence on Treatment Repetition Versus Potentiation

“…ATCC rigorously authenticates its cell lines using standardized methods described in its cell biology program documentation (http://www.atcc.org). An optically reporting A‐431/G4 clonal cell line expressing a red fluorescent protein and luciferase was developed using lentiviral methods . Cell line maintenance conditions have previously been described elsewhere …”

“…Synthesis and characterization of Ac‐sPPp (see Fig. a) has previously been described . Dynamic light scattering (DLS) was used to measure particle size distributions of Ac‐sPPp solutions (Zetasizer Nano ZS; Malvern Instruments, Inc., Southborough, MA).…”

“…For the luciferase‐based phototoxicity assay, all treatment conditions were similar to the MTT‐based assay, except cells were grown and PDT treated in 96‐well black‐welled/clear‐bottom plates. Viability was assayed 12–24 h post‐PDT by incubating cells with 0.15 mg mL −1 luciferin in complete culture medium for 5 min at 37°C and then immediately imaging plates on a custom‐built optical imager . Wells containing untreated cells served as control comparisons.…”

“…A‐431 or optically reporting A‐431/G4 tumors were implanted subcutaneously or intradermally in the pectoral or pelvic regions for imaging and PDT experiments. Tumor inoculation, tumor measurement, and anesthesia methods have been previously described . All animal experimentation was approved by Dartmouth College's Institutional Animal Care and Use Committee.…”

“…In our present study of the EGFR‐overexpressing A‐431 human vulvar squamous cell carcinoma, which shows exceptional radioresistance and exhibits high recurrence in immunocompromised mouse models, we found that passively targeted Ac‐sPPp PDT provides superior tumor control compared to PPa PDT and equivalent or better tumor control compared to anti‐EGFR Ac‐sPPp‐cetuximab PIC‐targeted PDT or Ac‐sPPp + Doxil PDT combination therapy. We also observed strong vascular PDT effects, regardless of whether Ac‐sPPp was targeted passively as a micellar solution or actively as a PIC.…”