Intratumoral heterogeneity within individual breast tumors is a well-known phenomenon that may contribute to drug resistance. This heterogeneity is dependent on several factors, such as types of oncogenic drivers and tumor precursor cells. The purpose of our study was to engineer a mouse mammary tumor model with intratumoral heterogeneity by using defined genetic perturbations. To achieve this, we used mice with knockout (−/−) of Ink4a/Arf, a tumor suppressor locus; these mice are known to be susceptible to non-mammary tumors such as fibrosarcoma. To induce mammary tumors, we retrovirally introduced an oncogene, HRAS(G12V), into Ink4a/Arf−/− mammary cells in vitro, and those cells were inoculated into syngeneic mice mammary fat pads. We observed 100% tumorigenesis. The tumors formed were negative for estrogen receptor, progesterone receptor, and HER2. Further, they had pathological features similar to those of human triple-negative breast cancer (e.g. pushing borders, central necrosis). The tumors were found to be heterogeneous and included two subpopulations: CD49f− quiescent cells and CD49f+ cells. Contrary to our expectation, CD49f− quiescent cells had high tumor-initiating potential and CD49f+ cells had relatively low tumor-initiating potential. Gene expression analysis revealed that CD49f− quiescent cells overexpressed epithelial-to-mesenchymal transition-driving genes, reminiscent of tumor-initiating cells and claudin-low breast cancer. Our animal model with intratumoral heterogeneity, derived from defined genetic perturbations, allows us to test novel molecular targeted drugs in a setting that mimics the intratumoral heterogeneity of human triple-negative breast cancer.

Introduction: Mammography is the most basic modality in breast cancer imaging. However, the overlap of breast tissue depicted on conventional two-dimensional mammography (2DMMG) may create significant obstacles to detecting abnormalities, especially in dense or heterogeneously dense breasts. In three-dimensional digital breast tomosynthesis (3DBT), tomographic images of the breast are reconstructed from multiple projections acquired at different angles. It has reported that this technology allows the generation of 3D data, therefore overcoming the limitations of conventional 2DMMG for Western women. We assessed the detectability of lesions by conventional 2DMMG and 3DBT in diagnosis of breast cancer for Japanese women. Methods: The subjects were 195 breasts of 99 patients (median age of 48 years, range 34~82 years) that had been pathologically diagnosed with breast cancer from December 20, 2010 through March 31, 2011. Both conventional 2DMMG and 3DBT imaging were performed for all patients. Detectability of lesions was assessed based on differences in category class. Results: Of the affected breasts, 77 (75.5%) had lesions assigned to the same categories by 2DMMG and 3DBT. For 24 (23.5%) lesions, the category increased in 3DBT indicating improvement in diagnostic performance compared to 2DMMG. 3DBT improved diagnostic sensitivity for patients with mass, focal asymmetric density (FAD), and architectural distortion. However, 3DBT was not statistically superior in diagnosis of the presence or absence of calcification. Conclusions: In this study, 3DBT was superior in diagnosing lesions in form of mass, FAD, and/or architectural distortion. 3DBT is a novel technique that may provide a breakthrough in solving the difficulties of diagnosis caused by parenchyma overlap for Japanese women.

Phyllodes tumors (PTs) are rare fibroepithelial tumors of the breast with epithelial and stromal components, and surgical resection is the standard and only available treatment for malignant PTs. To provide a better understanding of these tumors, we developed mouse models that recapitulate the pathological and clinical properties of human malignant PTs. Mouse undifferentiated mammary gland cells were infected with a retrovirus encoding the human oncoprotein H-Ras G12V , and the infected cells were transplanted orthotopically into the mammary fat pads of syngeneic mice. The transplanted cells showed a high tumorigenic activity, with the resulting tumors manifesting pathological characteristics including stromal overgrowth similar to those of human malignant PTs. The tumors also showed high rates of both local recurrence and lung metastasis. Our models may prove useful for studies of the pathophysiology of malignant PTs as well as facilitate the development of new treatments.

Although 68.2 % of ILC patients were evaluated "appropriately" even after PST, higher additional total mastectomy rates should be considered when selecting the surgical procedure.

Breast cancer is one of the leading causes of death in women worldwide. To develop novel therapeutic approaches for the refractory cases, the mouse models which recapitulate the tumor tissues biologically and pathologically similar to human breast cancer are required. Although xenograft models of established cell lines in immune-deficient mice are frequently used for preclinical experiments, such xenograft models are not sufficient because the heterogenous structure based on the microenvironment and the intrinsic characteristics of cancer cells is not correctly formed. In this study, we have established induced cancer stem cells (iCSC) from normal mouse mammary stem/progenitor cells through minimal required genetic manipulations and generated mouse breast cancer models by inoculating the iCSCs in the mammary fat pads. Initially, we established iCSC by introducing the H-RasV12 into Ink4a/Arf-knockout mammary stem/progenitor cells and this iCSC formed tumor similar to human triple negative breast cancer in mouse. This finding suggested that two genetic events, an activation of oncogenic signal and a tumor suppressor inactivation, are required for generating the breast cancer iCSC. Anaplastic Lymphoma Kinase (ALK) gene, which encodes a receptor tyrosine kinase, was reported to be amplified and/or overexpressed up to 86% of patients in inflammatory breast cancer, and pleiotrophin (PTN), which is a physiological ligand for ALK, was also shown to be highly expressed in about 60% of human breast cancers. Therefore, we hypothesized that ALK pathway is involved in tumorigenesis of breast cancers and, then, attempted to generate iCSC by using ALK gene. Interestingly, we found that one of the naturally occurring mutations of ALK is sufficient for generating iCSC and tumor formation in vivo without any prior tumor suppressor inactivation. The ALK-induced iCSCs developed highly aggressive breast cancers in mice. Furthermore, the tumor formation was significantly suppressed when the ALK-induced iCSCs were generated by using mammary stem/progenitor cells derived from mouse deficient in CD44 which is a CSC marker. We have recently revealed a role of CD44, in particular that of a variant isoform (CD44v), in the protection of CSCs from high levels of oxidative stress derived from both tumor cells and their microenvironment (Cancer Cell 19: 387–400, 2011; Cancer Res 72: 1438–1448, 2012; Nat Commun 3: 883, 2012). We will discuss the underlying mechanism of ALK-induced tumorigenesis and a role of CD44 in the CSC functions. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-04-01.