A thermal "window" of immunogenic cell death (ICD) elicited by nanoparticle-based photothermal therapy (PTT) in an animal model of neuroblastoma is described. In studies using Prussian blue nanoparticles to administer photothermal therapy (PBNP-PTT) to established localized tumors in the neuroblastoma model, it is observed that PBNP-PTT conforms to the "more is better" paradigm, wherein higher doses of PBNP-PTT generates higher cell/local heating and thereby more cell death, and consequently improved animal survival. However, in vitro analysis of the biochemical correlates of ICD (ATP, high-motility group box 1, and calreticulin) elicited by PBNP-PTT demonstrates that PBNP-PTT triggers a thermal window of ICD. ICD markers are highly expressed within an optimal temperature (thermal dose) window of PBNP-PTT (63.3-66.4 °C) as compared with higher (83.0-83.5 °C) and lower PBNP-PTT (50.7-52.7 °C) temperatures, which both yield lower expression. Subsequent vaccination studies in the neuroblastoma model confirm the in vitro findings, wherein PBNP-PTT administered within the optimal temperature window results in long-term survival (33.3% at 100 d) compared with PBNP-PTT administered within the higher (0%) and lower (20%) temperature ranges, and controls (0%). The findings demonstrate a tunable immune response to heat generated by PBNP-PTT, which should be critically engaged in the administration of PTT for maximizing its therapeutic benefits.

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Purpose Our publications demonstrate that physiological concentrations of estrogen (E2) induce endoplasmic reticulum and oxidative stress which finally result in apoptosis in E2-deprived breast cancer cells, MCF-7:5C. c-Src is involved in the process of E2-induced stress. To mimic the clinical administration of c-Src inhibitors, we treated cells with either E2, a c-Src inhibitor PP2, or the combination for 8 weeks to further explore the apoptotic potential of the c-Src inhibitor and E2 on MCF-7:5C cells. Methods Protein levels of receptors and signaling pathways were examined by immunoblotting. Expression of mRNA was detected through real-time PCR. Cell cycles were analyzed by flow cytometry. Results Long-term treatment with PP2 alone or E2 alone decreased cell growth. In contrast, a combination of PP2 and E2 blocked apoptosis and the resulting cell line (MCF-7:PF) was unique, as they grew vigorously in culture with physiological levels of E2, which could be blocked by the pure antiestrogen ICI182,780. One major change was that PP2 collaborated with E2 to increase the level of insulin-like growth factor-1 receptor beta (IGF-1Rβ). Blockade of IGF-1Rβ completely abolished E2-stimulated growth in MCF-7:PF cells. Furthermore, combination treatment up-regulated transcription factors, Twist1 and Snail, and repressed E-cadherin expression which made MCF-7:PF cells display a characteristic phenotype of epithelial-mesenchymal transition (EMT). Conclusions These data illustrate the role of the c-Src inhibitor to block E2-induced apoptosis and enhance E2-stimulated growth. Caution must be exercised when considering c-Src inhibitors in clinical trials following the development of acquired resistance to aromatase inhibitors, especially in the presence of the patient’s own estrogen.

Models of long-term estrogen-deprived breast cancer cells are utilized in the laboratory to mimic clinical aromatase inhibitor-resistant breast cancer and serve as a tool to discover new therapeutic strategies. The MCF-7:5C and MCF-7:2A subclones were generated through long-term estrogen deprivation of estrogen receptor (ER)-positive MCF-7 cells, and represent anti-hormone-resistant breast cancer. MCF-7:5C cells paradoxically undergo estrogen-induced apoptosis within seven days of estrogen (estradiol, E2) treatment; MCF-7:2A cells also experience E2-induced apoptosis but evade dramatic cell death until approximately 14 days of treatment. To discover and define the mechanisms by which MCF-7:2A cells survive two weeks of E2 treatment, systematic experiments were performed in this study. The data suggest that MCF-7:2A cells employ stronger antioxidant defense mechanisms than do MCF-7:5C cells, and that oxidative stress is ultimately required for MCF-7:2A cells to die in response to E2 treatment. Tumor necrosis factor (TNF) family member activation is also essential for E2-induced apoptosis to occur in MCF-7:2A cells; upregulation of TNFα occurs simultaneously with oxidative stress activation. Although the unfolded protein response (UPR) signaling pattern is similar to that in MCF-7:5C cells, it is not sufficient to cause cell death in MCF-7:2A cells. Additionally, increased insulin-like growth factor receptor β (IGF-1Rβ) confers a mechanism of growth and anti-apoptotic advantage in MCF-7:2A cells.

Translational research for the treatment and prevention of breast cancer depends upon the four Ms: models, molecules, and mechanisms in order to create medicines. The process, to target the estrogen receptor (ER) in estrogen-dependent breast cancer, has yielded significant advances in patient survivorship and the first approved medicines (tamoxifen and raloxifene) to reduce the incidence of any cancer in high- or low-risk women. This review focuses on the critical role of the few ER-positive cell lines (MCF-7, T47D, BT474, ZR-75) that continue to advance our understanding of the estrogen-regulated biology of breast cancer. More importantly, the model cell lines have provided an opportunity to document the development and evolution of acquired antihormone resistance. The description of this evolutionary process that occurs in micrometastatic disease during up to a decade of adjuvant therapy would not be possible in the patient. The use of the MCF-7 breast cancer cell line in particular has been instrumental in discovering a vulnerability of ER-positive breast cancer exhaustively treated with antihormone therapy. Physiologic estradiol acts as an apoptotic trigger to cause tumor regression. These unanticipated findings in the laboratory have translated to clinical advances in our knowledge of the paradoxical role of estrogen in the life and death of breast cancer.

Photothermal therapy using CpG oligodeoxynucleotide-coated Prussian blue nanoparticles increases the tumor antigenicity and adjuvanticity, eliciting long-term tumor regression and immunological memory.

Hormone replacement therapy (HRT) is widely used to manage menopausal symptoms in women, and can comprise an estrogen alone or an estrogen combined with a progestin. The Women’s Health Initiative demonstrated in their randomized trials that estrogen alone HRT decreases the risk of breast cancer in post-menopausal women, while combined estrogen plus a progestin (medroxyprogesterone acetate, MPA) HRT increases this risk. Long-term estrogen-deprived MCF-7:5C cells were used to model the post-menopausal breast cancer cell environment. MPA is able to modify E2-induced apoptosis in MCF-7:5C cells. MPA, similar to dexamethasone (Dex) increases GR transcriptional activity, increases SGK1, a GR target gene, and can be blocked by RU486 (an antiglucocorticoid), suggesting it functions through the GR. Norethindrone acetate (NETA), another progestin used in HRT, acts like an estrogen at high doses, up-regulating ER-target genes and generating apoptosis in MCF-7:5C cells. The data suggests that women taking HRT comprising an estrogen plus MPA may have an increased the risk of breast cancer due to MPA acting as a glucocorticoid and blunting E2-induced apoptosis in this environment. Therefore, perhaps other approved progestins (e.g. NETA) should be considered as alternatives to MPA.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive tumors with low survival rates and the leading cause of death in neurofibromatosis type 1 (NF1) patients under 40 years old. Surgical resection is the standard of care for MPNSTs, but is often incomplete and can generate loss of function, necessitating the development of novel treatment methods for this patient population. Here, we describe a novel combination therapy comprising MEK inhibition and nanoparticle-based photothermal therapy (PTT) for MPNSTs. MEK inhibitors block activity driven by Ras, an oncogene constitutively activated in NF1-associated MPNSTs, while PTT serves as a minimally invasive method to ablate cancer cells. Our rationale for combining these seemingly disparate techniques for MPNSTs is based on several reports demonstrating the efficacy of systemic chemotherapy with local PTT. We combine the MEK inhibitor, PD-0325901 (PD901), with Prussian blue nanoparticles (PBNPs) as PTT agents, to block MEK activity and simultaneously ablate MPNSTs. Our data demonstrate the synergistic effect of combining PD901 with PBNP-based PTT, which converge through the Ras pathway to generate apoptosis, necrosis, and decreased proliferation, thereby mitigating tumor growth and increasing survival of MPNST-bearing animals. Our results suggest the potential of this novel local-systemic combination “nanochemotherapy” for treating patients with MPNSTs.