Background: PTEN-deficient tumors are dependent on PI3Kβ activity, making PI3Kβ a compelling target. We evaluated the efficacy of PI3Kβ inhibitor AZD8186 on tumors with PTEN loss. Results: In vitro cell viability assay and immunoblotting demonstrated that PTEN loss was significantly correlated with AZD8186 sensitivity in triple negative breast cancer (TNBC) cell lines. Colony formation assay confirmed sensitivity of PTENdeficient cell lines to AZD8186. AZD8186 inhibited PI3K signaling in PTEN loss TNBC cells. AZD8186 in combination with paclitaxel, eribulin had synergistic effects on growth inhibition in PTEN loss cells. AZD8186 promoted apoptosis in PTEN loss cells which was synergized by paclitaxel. In vivo, AZD8186 had limited activity as a single agent, but enhanced antitumor activity when combined with paclitaxel in MDA-MB-436 and MDA-MB-468 cell-line xenografts. AZD8186 significantly enhanced antitumor efficacy of anti-PD1 antibodies in the PTEN-deficient BP murine melanoma xenograft model, but not in the PTEN-wild-type CT26 xenograft model. Methods: In vitro, cell proliferation and colony formation assays were performed to determine cell sensitivity to AZD8186. Immunoblotting was performed to assess PTEN expression and PI3K signaling activity. FACS was performed to evaluate apoptosis. In vivo, antitumor efficacy of AZD8186 and its combinations were evaluated. Conclusions: AZD8186 has single agent efficacy in PTEN-deficient TNBC cell lines in vitro, but has limited single agent efficacy in vivo. However, AZD8186 has enhanced efficacy when combined with paclitaxel and anti-PD1 in vivo. Further study is needed to determine optimal combination therapies for PTEN-deficient solid tumors.

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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.

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A novel pyropheophorbide-a (PPa) derivative, Ac-sPPp, was developed in our lab for targeted photodynamic therapy (PDT) and combination therapies. Its versatile peptide moiety, high water-solubility, amphiphilicity, and micellar aggregation allow efficient coupling to targeting moieties and convenient mixing with other therapeutics. Photosensitizer immunoconjugate (PIC) targeted PDT, using Ac-sPPp conjugated to therapeutic anti-epidermal growth factor receptor (EGFR) antibody cetuximab, and PDT + chemotherapy combination treatment, using Ac-sPPp mixed with stealth liposomal doxorubicin (Doxil), were investigated as promising strategies for potentiating PDT and improving target specificity. Passively targeted PDT with Ac-sPPp only or surfactant-solubilized PPa was also investigated for comparison. The A-431 human vulvar squamous cell carcinoma, xenografted in nude mice, was chosen as a tumor model because of its high EGFR expression and sensitivity to liposomal doxorubicin in vitro. Fluorescence imaging and PDT experiments showed that Ac-sPPp formulations circulated far longer and provided superior tumor contrast and superior tumor control compared to PPa. Strong PDT vascular effects were observed by laser Doppler imaging regardless of whether Ac-sPPp was passively or actively targeted. Passively targeted Ac-sPPp PDT gave equivalent or better tumor control than PIC-targeted PDT or PDT + Doxil combination therapy, and when treatments were repeated, it also yielded the highest cure rate.