Karen M. Bussard

The bone microenvironment in metastasis; what is special about bone?

The skeleton is a common destination for many cancer metastases including breast and prostate cancer. There are many characteristics of bone that make it an ideal environment for cancer cell migration and colonization. Metaphyseal bone, found at the ends of long bone, in ribs, and in vertebrae, is comprised of trabecular bone interspersed with marrow and rich vasculature. The specialized microvasculature is adapted for the easy passage of cells in and out of the bone marrow. Moreover, the metasphyseal regions of bone are constantly undergoing remodeling, a process that releases growth factors from the matrix. Bone turnover also involves the production of numerous cytokines and chemokines that provide a means of communication between osteoblasts and osteoclasts, but co-incidentally can also attract and support metastatic cells. Once in the marrow, cancer cells can interact directly and indirectly with osteoblasts and osteclasts, as well as hematopoietic and stromal cells. Cancer cells secrete factors that affect the network of cells in the bone microenvironment as well as interact with other cytokines. Additionally, transient cells of the immune system may join the local mileau to ultimately support cancer cell growth. However, most metastasized cells that enter the bone marrow are transient; a few may remain in a dormant state for many years. Advances in understanding the bone cell-tumor cell interactions are key to controlling, if not preventing metastasis to bone.

Tumor-associated stromal cells as key contributors to the tumor microenvironment.

The tumor microenvironment is a heterogeneous population of cells consisting of the tumor bulk plus supporting cells. It is becoming increasingly evident that these supporting cells are recruited by cancer cells from nearby endogenous host stroma and promote events such as tumor angiogenesis, proliferation, invasion, and metastasis, as well as mediate mechanisms of therapeutic resistance. In addition, recruited stromal cells range in type and include vascular endothelial cells, pericytes, adipocytes, fibroblasts, and bone-marrow mesenchymal stromal cells. During normal wound healing and inflammatory processes, local stromal cells change their phenotype to become that of reactive stroma. Under certain conditions, however, tumor cells can co-opt these reactive stromal cells and further transition them into tumor-associated stromal cells (TASCs). These TASCs express higher levels of proteins, including alpha-smooth muscle actin, fibroblast activating protein, and matrix metalloproteinases, compared with their normal, non-reactive counterparts. TASCs are also known to secrete many pro-tumorigenic factors, including IL-6, IL-8, stromal-derived factor-1 alpha, vascular endothelial growth factor, tenascin-C, and matrix metalloproteinases, among others, which recruit additional tumor and pro-tumorigenic cells to the developing microenvironment. Here, we review the current literature pertaining to the origins of recruited host stroma, contributions toward tumor progression, tumor-associated stromal cells, and mechanisms of crosstalk between endogenous host stroma and tumor cells.

Reprogramming human cancer cells in the mouse mammary gland.

The tissue microenvironment directs stem/progenitor cell behavior. Cancer cells are also influenced by the microenvironment. It has been shown that, when placed into blastocysts, cancer cells respond to embryonic cues and differentiate according to the tissue type encountered during ontological development. Previously, we showed that the mouse mammary gland was capable of redirecting adult mouse testicular and neural stem/progenitor cells toward a mammary epithelial cell fate during gland regeneration. Here, we report that human embryonal carcinoma cells proliferate and produce differentiated mammary epithelial cell progeny when mixed with mouse mammary epithelial cells and inoculated into the epithelium-free mammary fat pads of athymic nude mice. Fluorescence in situ hybridization confirmed the presence of human cell progeny in the mammary outgrowths for human centromeric DNA, as well as immunochemistry for human-specific breast epithelial cytokeratins and human-specific milk proteins in impregnated transplant hosts. It was found that the number of human cells increased by 66- to 660-fold during mammary epithelial growth and expansion as determined by human cytokeratin expression. All features found in primary outgrowths were recapitulated in the secondary outgrowths from chimeric implants. These results show that human embryonal carcinoma-derived progeny interact with mouse mammary cells during mammary gland regeneration and are directed to differentiate into cells that exhibit diverse mammary epithelial cell phenotypes. This is the first demonstration that human cells are capable of recognizing the signals generated by the mouse mammary gland microenvironment present during gland regeneration in vivo.

Osteoblasts Are a Major Source of Inflammatory Cytokines in the Tumor Microenvironment of Bone Metastatic Breast Cancer

Metastatic breast cancer cells co‐opt the cells of the bone to increase their production of inflammatory cytokines. Here, we sought to identify key cytokines expressed by osteoblasts in vitro and in vivo in the presence of MDA‐MB‐231 metastatic breast cancer cells, including a bone‐seeking variant. We hypothesized that osteoblast‐derived cytokines increase in the presence of metastatic breast cancer cell conditioned medium (CM), act as chemoattractants for cancer cells, and enhance osteoclast formation. We detected increases in the concentrations of osteoblast‐derived IL‐6, MCP‐1, VEGF, MIP‐2, and KC in vitro in culture supernatants from MC3T3‐E1 cells in the presence of metastatic breast cancer cell CM and from cancer‐bearing femurs ex vivo. A comparison of cancer cell‐ and osteoblast‐derived cytokines revealed that while breast cancer cells expressed the same or equivalent cytokines as the osteoblasts, the breast cancer cells only produced picogram quantities of MCP‐1; osteoblasts expressed nanogram amounts. Bone‐derived MCP‐1 increased in the proximal metaphysis, an area where breast cancer cells preferentially trafficked following intracardiac inoculation in athymic mice. An MDA‐MB‐231 bone‐seeking variant was not different from parental lines. Osteoblast CM was a potent chemoattractant for metastatic breast cancer cells. Furthermore, culture supernatants of osteoblasts treated with breast cancer cell CM enhanced osteoclast formation. These findings suggest that bone metastatic breast cancer cells utilize osteoblast‐derived cytokines to facilitate breast cancer cell colonization and survival upon arrival in the bone microenvironment. J. Cell. Biochem. 111: 1138–1148, 2010.

Human Breast Cancer Cells Are Redirected to Mammary Epithelial Cells upon Interaction with the Regenerating Mammary Gland Microenvironment In-Vivo

Breast cancer is the second leading cause of cancer deaths in the United States. At present, the etiology of breast cancer is unknown; however the possibility of a distinct cell of origin, i.e. a cancer stem cell, is a heavily investigated area of research. Influencing signals from the tissue niche are known to affect stem cells. Literature has shown that cancer cells lose their tumorigenic potential and display ‘normal’ behavior when placed into ‘normal’ ontogenic environments. Therefore, it may be the case that the tissue microenvironment is able to generate signals to redirect cancer cell fate. Previously, we showed that pluripotent human embryonal carcinoma cells could be redirected by the regenerating mammary gland microenvironment to contribute epithelial progeny for ‘normal’ gland development in-vivo. Here, we show that that human metastatic, non-metastatic, and metastasis-suppressed breast cancer cells proliferate and contribute to normal mammary gland development in-vivo without tumor formation. Immunochemistry for human-specific mitochondria, keratin 8 and 14, as well as human-specific milk proteins (alpha-lactalbumin, impregnated transplant hosts) confirmed the presence of human cell progeny. Features consistent with normal mammary gland development as seen in intact hosts (duct, lumen formation, development of secretory acini) were recapitulated in both primary and secondary outgrowths from chimeric implants. These results suggest the dominance of the tissue microenvironment over cancer cell fate. This work demonstrates that cultured human breast cancer cells (metastatic and non-metastatic) respond developmentally to signals generated by the mouse mammary gland microenvironment during gland regeneration in-vivo.

The Mammary Gland Microenvironment Directs Progenitor Cell Fate In Vivo

The mammary gland is a unique organ that continually undergoes postnatal developmental changes. In mice, the mammary gland is formed via signals from terminal end buds, which direct ductal growth and elongation. Intriguingly, it is likely that the entire cellular repertoire of the mammary gland is formed from a single antecedent cell. Furthermore, in order to produce progeny of varied lineages (e.g., luminal and myoepithelial cells), signals from the local tissue microenvironment influence mammary stem/progenitor cell fate. Data have shown that cells from the mammary gland microenvironment reprogram adult somatic cells from other organs (testes, nerve) into cells that produce milk and express mammary epithelial cell proteins. Similar results were found for human tumorigenic epithelial carcinoma cells. Presently, it is unclear how the deterministic power of the mammary gland microenvironment controls epithelial cell fate. Regardless, signals generated by the microenvironment have a profound influence on progenitor cell differentiation in vivo.

Localization of osteoblast inflammatory cytokines MCP-1 and VEGF to the matrix of the trabecula of the femur, a target area for metastatic breast cancer cell colonization

Bone likely provides a hospitable environment for cancer cells as suggested by their preferential localization to the skeleton. Previous work has shown that osteoblast-derived cytokines increased in the presence of metastatic breast cancer cells. Thus, we hypothesized that osteoblast-derived cytokines, in particular IL-6, MCP-1, and VEGF, would be localized to the bone metaphyses, an area to which breast cancer cells preferentially traffic. Human metastatic MDA-MB-231 breast cancer cells were inoculated into the left ventricle of the heart of athymic mice. Three to four weeks later, tumor localization within isolated femurs was examined using μCT and MRI. In addition, IL-6, MCP-1, and VEGF localization were assayed via immunohistochemistry. We found that MDA-MB-231 cells colonized trabecular bone, the area in which murine MCP-1 and VEGF were visualized in the bone matrix. In contrast, IL-6 was expressed by murine cells throughout the bone marrow. MDA-MB-231 cells produced VEGF, whose expression was not only associated with the breast cancer cells, but also increased with tumor growth. This is the first study to localize MCP-1, VEGF, and IL-6 in bone compartments via immunohistochemistry. These data suggest that metastatic cancer cells may co-opt bone cells into creating a niche facilitating cancer cell colonization.

Immortalized, premalignant epithelial cell populations contain long-lived, label-retaining cells that asymmetrically divide and retain their template DNA

IntroductionDuring selective segregation of DNA, a cell asymmetrically divides and retains its template DNA. Asymmetric division yields daughter cells whose genome reflects that of the parents, simultaneously protecting the parental cell from genetic errors that may occur during DNA replication. We hypothesized that long-lived epithelial cells are present in immortal, premalignant cell populations, undergo asymmetric division, retain their template DNA strands, and cycle both during allometric growth and during pregnancy.MethodsThe glands of 3-week-old immune-competent Balb/C female mice were used intact or cleared of host epithelium and implanted with ductal-limited, lobule-limited, or alveolar-ductal progenitor cells derived from COMMA-D1 pre-malignant epithelial cells. 5-Bromo-2-deoxyuridine (5-BrdU) was administered to identify those cells that retain their template DNA. Nulliparous mice were then either injected with [3H]-thymidine (3H-TdR) to distinguish 5-BrdU label-retaining cells that enter the cell cycle and euthanized, or mated, injected with 3H-TdR, and euthanized at various days after coitus. Sections were stained for estrogen receptor-α (ER-α) or progesterone receptor (PR) with immunohistochemistry. Cells labeled with both 5-BrdU and 3H-TdR were indicative of label-retaining epithelial cells (LRECs).ResultsCells that retained a 5-BrdU label and cells labeled with [3H]-thymidine were found in all mice and were typically detected along the branching epithelium of mature mouse mammary glands. Cells containing double-labeled nuclei (LRECs) were found in the intact mammary glands of both pregnant and nulliparous mice, and in mammary glands implanted with premalignant cells. Double-labeled cells (3H-TdR/5-BrdU) represent a small portion of cells in the mammary gland that cycle and retain their template DNA (5-BrdU). Some label-retaining cells were also ER-α or PR positive. LRECs distributed their second label (3H-TdR) to daughter cells, and this effect persisted during pregnancy. LRECs, and small focal hyperplasia, were found in all immortalized premalignant mammary-implant groups.ConclusionsThe results indicate that a subpopulation of long-lived, label-retaining epithelial cells (LRECs) is present in immortal premalignant cell populations. These LRECs persist during pregnancy, retain their original DNA, and a small percentage express ER-α and PR. We speculate that LRECs in premalignant hyperplasia represent the long-lived (memory) cells that maintain these populations indefinitely.

Ex-vivo Analysis of the Bone Microenvironment in Bone Metastatic Breast Cancer

In humans, breast cancer has a predilection to metastasize to the skeleton. While the mechanism for preferential metastasis is unknown, the bone microenvironment likely provides a fertile soil for metastatic breast cancer cells. In order to examine the bone microenvironment ex-vivo following the formation of breast cancer metastases, several techniques may be employed: fluorescence stereomicroscopy, magnetic resonance imaging (MRI), microCT (μCT), immunohistochemistry, and cytokine arrays, to name a few. These methods allow for a comprehensive evaluation of the bone microenvironment during bone metastatic breast cancer. By identifying alterations in the bone niche caused by metastatic breast cancer cells, it may be possible to block or disrupt these factors through the use of targeted drugs. Appropriate therapeutic treatment would allow for an improved quality of life and longer survival time for individuals with bone metastatic breast cancer.

Understanding Mitochondrial Polymorphisms in Cancer

Alterations in mitochondrial DNA (mtDNA) were once thought to be predominantly innocuous to cell growth. Recent evidence suggests that mtDNA undergo naturally occurring alterations, including mutations and polymorphisms, which profoundly affect the cells in which they appear and contribute to a variety of diseases, including cardiovascular disease, diabetes, and cancer. Furthermore, interplay between mtDNA and nuclear DNA has been found in cancer cells, necessitating consideration of these complex interactions for future studies of cancer mutations and polymorphisms. In this issue of Cancer Research, Vivian and colleagues utilize a unique mouse model, called Mitochondrial Nuclear eXchange mice, that contain the nuclear DNA from one inbred mouse strain, and the mtDNA from a different inbred mouse strain to examine the genome-wide nuclear DNA methylation and gene expression patterns of brain tissue. Results demonstrated there were alterations in nuclear DNA expression and DNA methylation driven by mtDNA. These alterations may impact disease pathogenesis. In light of these results, in this review, we highlight alterations in mtDNA, with a specific focus on polymorphisms associated with cancer susceptibility and/or prognosis, mtDNA as cancer biomarkers, and considerations for investigating the role of mtDNA in cancer progression for future studies. Cancer Res; 77(22); 6051-9. ©2017 AACR.