Purpose: Breast cancer stem cells have been shown to be associated with resistance to chemotherapy in vitro, but their clinical significance remains to be clarified. The aim of this study was to investigate whether cancer stem cells were clinically significant for resistance to chemotherapy in human breast cancers. Experimental Design: Primary breast cancer patients (n = 108) treated with neoadjuvant chemotherapy consisting of sequential paclitaxel and epirubicin-based chemotherapy were included in the study. Breast cancer stem cells were identified by immunohistochemical staining of CD44/CD24 and aldehyde dehydrogenase 1 (ALDH1) in tumor tissues obtained before and after neoadjuvant chemotherapy. CD44 + /CD24-tumor cells or ALDH1-positive tumor cells were considered stem cells. Results: Thirty (27.8%) patients achieved pathologic complete response (pCR). ALDH1-positive tumors were significantly associated with a low pCR rate (9.5% versus 32.2%; P = 0.037), but there was no significant association between CD44 + /CD24 -tumor cell proportions and pCR rates. Changes in the proportion of CD44 + /CD24 -or ALDH1-positive tumor cells before and after neoadjuvant chemotherapy were studied in 78 patients who did not achieve pCR. The proportion of ALDH1-positive tumor cells increased significantly (P < 0.001) after neoadjuvant chemotherapy, but that of CD44 + /CD24 -tumor cells did not. Conclusions: Our findings suggest that breast cancer stem cells identified as ALDH1-positive, but not CD44

Recently, aldehyde dehydrogenase (ALDH) 1 has been identified as a reliable marker for breast cancer stem cells. E vidence has recently been accumulating to support the cancer stem cell hypothesis for solid tumors, including breast cancer, which holds that cancers are driven by a small subpopulation of stem cells that are capable of self-renewal and give rise to multipotent progenitor cells that ultimately differentiate into all cell types within the tumor.(1) Al-Hajj et al. were the first to distinguish tumorigenic from non-tumorigenic cancer cells by using the cell surface markers CD44 and CD24.(2) They have shown that cancer stem cells in a population of tumor cells are enriched with the CD44 + and CD24 -phenotype because as few as 100 tumor cells with this phenotype were able to produce tumors in immunodeficient mice, whereas tumor cells with other CD44 and CD24 phenotypes were unable or rarely able to produce tumors even when as many as 10 5 -10 6 tumor cells were inoculated into such mice. + cancer stem cells may well be clinically useful for patient prognosis. In the study reported here, we therefore investigated the clinicopathological characteristics of breast cancers with ALDH1 + cancer stem cells and also compared ALDH1 expression in primary tumors and axillary metastases. In addition, the ER, Ki67, and HER2 status of ALDH1 + tumor cells was investigated on a cell-by-cell basis by means of double immunohistochemical staining for further characterization of the phenotype of breast cancer stem cells. Materials and MethodsPatients and breast tumor tissues. Tumor tissue samples were obtained from 203 primary breast cancer patients (mean age, 52.6 years; range, 32-86 years) who underwent mastectomy or breast-conserving surgery between January 1993 and December 1997 at Osaka University Hospital, Osaka, Japan. Tumor tissues were fixed in 10% buffered formalin and embedded in paraffin. This study protocol was approved by the Ethics Committee of Osaka University.For adjuvant therapy, 85 patients were treated with hormonal therapy (tamoxifen, n = 74; toremifene, n = 7; gosereline, n = 3; or gosereline + tamoxifen, n = 1), 22 with chemotherapy (fluoropyrimidine, n = 8; cyclophosphamide + methotrexate + 5-fluorouracil, n = 7; cyclophosphamide + adriamycin + 5-fluorouracil, n = 4; or high-dose chemotherapy, n = 3), and 84 with chemohormonal therapy (fluoropyrimidine, n = 37; cyclophosphamide + methotrexate + 5-fluorouracil, n = 25; cyclophosphamide + adriamycin + 5-fluorouracil, n = 20; or high-dose chemotherapy, n = 2; plus tamoxifen, n = 78; toremifene, n = 1; gosereline, n = 3; or gosereline + tamoxifen, n = 2). Twelve patients received no adjuvant therapy.

Current treatments for liver metastases arising from primary breast and lung cancers are minimally effective. One reason for this unfavorable outcome is that liver metastases are poorly vascularized, limiting the ability to deliver therapeutics from the systemic circulation to lesions. Seeking to enhance transport of agents into the tumor microenvironment, we designed a system in which nanoparticle albumin-bound paclitaxel (nAb-PTX) is loaded into a nanoporous solid multistage nanovector (MSV) to enable the passage of the drug through the tumor vessel wall and enhance its interaction with liver macrophages. MSV enablement increased nAb-PTX efficacy and survival in mouse models of breast and lung liver metastasis. MSV-nAb-PTX also augmented the accumulation of PTX and MSV in the liver, specifically in macrophages, whereas PTX levels in the blood were unchanged after administering MSV-nAb-PTX or nAb-PTX. In vitro studies demonstrated that macrophages treated with MSV-nAb-PTX remained viable and were able to internalize, retain, and release significantly higher quantities of PTX compared to treatment with nAb-PTX. The cytotoxic potency of the released PTX was also confirmed in tumor cells cultured with the supernatants of macrophage treated with MSV-nAB-PTX. Collectively, our findings showed how redirecting nAb-PTX to liver macrophages within the tumor microenvironment can elicit a greater therapeutic response in patients with metastatic liver cancer, without increasing systemic side-effects.

Hypovascularization in tumors such as liver metastases originating from breast and other organs correlates with poor chemotherapeutic response and higher mortality. Poor prognosis is linked to impaired transport of both low- and high-molecular weight drugs into the lesions and to high washout rate. Nanoparticle albumin-bound-paclitaxel (nAb-PTX) has demonstrated benefits in clinical trials when compared to paclitaxel and docetaxel. However, its therapeutic efficacy for breast cancer liver metastasis is disappointing. As macrophages are the most abundant cells in the liver tumor microenvironment, we design a multistage system employing macrophages to deliver drugs into hypovascularized metastatic lesions, and perform in vitro, in vivo, and in silico evaluation. The system encapsulates nAb-PTX into nanoporous biocompatible and biodegradable multistage vectors (MSV), thus promoting nAb-PTX retention in macrophages. We develop a 3D in-vitro model to simulate clinically observed hypo-perfused tumor lesions surrounded by macrophages. This model enables evaluation of nAb-PTX and MSV-nab PTX efficacy as a function of transport barriers. Addition of macrophages to this system significantly increases MSV-nAb-PTX efficacy, revealing the role of macrophages in drug transport. In the in vivo model, a significant increase in macrophage number, as compared to unaffected liver, is observed in mice, confirming the in vitro findings. Further, a mathematical model linking drug release and retention from macrophages is implemented to project MSV-nAb-PTX efficacy in a clinical setting. Based on macrophage presence detected via liver tumor imaging and biopsy, the proposed experimental/computational approach could enable prediction of MSV-nab PTX performance to treat metastatic cancer in the liver.

The capillary wall is the chief barrier to tissue entry of therapeutic nanoparticles, thereby dictating their efficacy. Collagen fibers are an important component of capillary walls, affecting leakiness in healthy or tumor vasculature. Using a computational model along with in vivo systems, we compared how collagen structure affects the diffusion flux of a 1 nm chemotherapeutic molecule (doxorubicin [DOX]) and an 80 nm chemotherapy-loaded pegylated liposome (DOX-PLD) in tumor vasculature. We found a direct correlation between the collagen content around a tumor vessel to the permeability of that vessel permeability to DOX-PLD, indicating that collagen content may offer a biophysical marker of extravasation potential of liposomal drug formulations. Our results also suggested that while pharmacokinetics determined the delivery of DOX and DOX-PLD to the same tumor phenotype, collagen content determined the extravasation of DOX-PLD to different tumor phenotypes. Transport physics may provide a deeper view into how nanotherapeutics cross biological barriers, possibly helping explain the balance between biological and physical aspects of drug delivery.