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Benign mammary epithelial cells enhance the transformed phenotype of human breast cancer cells.

Poczobutt JM, Tentler J, Lu X, Schedin PJ, Gutierrez-Hartmann A - BMC Cancer (2010)

Bottom Line: Conditioned media derived from G2B-10A cells enhanced colony formation of R2-T1AS cells, whereas prior paraformaldehyde (PFA) fixation of G2B-10A cells abrogated this enhancement effect.These results indicate that soluble factors secreted by G2B-10A cells play a less important role in promoting R2-T1AS tumorigenesis in vivo, and that additional components are operative in the nude mouse xenograft assay.Finally, using array analysis, we found that both live and PFA-fixed G2B-10A cells induced R2-T1AS cells to secrete specific cytokines (IL-6 and GM-CSF), suggesting that cell-cell contact activates R2-T1AS cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biology Program, University of Colorado Denver, Aurora, CO 80045, USA.

ABSTRACT

Background: Recent research has yielded a wealth of data underscoring the key role of the cancer microenvironment, especially immune and stromal cells, in the progression of cancer and the development of metastases. However, the role of adjacent benign epithelial cells, which provide initial cell-cell contacts with cancer cells, in tumor progression has not been thoroughly examined. In this report we addressed the question whether benign MECs alter the transformed phenotype of human breast cancer cells.

Methods: We used both in vitro and in vivo co-cultivation approaches, whereby we mixed GFP-tagged MCF-10A cells (G2B-10A), as a model of benign mammary epithelial cells (MECs), and RFP-tagged MDA-MB-231-TIAS cells (R2-T1AS), as a model of breast cancer cells.

Results: The in vitro studies showed that G2B-10A cells increase the colony formation of R2-T1AS cells in both soft agar and clonogenicity assays. Conditioned media derived from G2B-10A cells enhanced colony formation of R2-T1AS cells, whereas prior paraformaldehyde (PFA) fixation of G2B-10A cells abrogated this enhancement effect. Moreover, two other models of benign MECs, MCF-12A and HuMECs, also enhanced R2-T1AS colony growth in soft agar and clonogenicity assays. These data reveal that factors secreted by benign MECs are responsible for the observed enhancement of the R2-T1AS transformed phenotype. To determine whether G2B-10A cells enhance the tumorigenic growth of co-injected R2-T1AS cells in vivo, we used the nude mouse xenograft assay. Co-injecting R2-T1AS cells with G2B-10A cells +/- PFA-fixation, revealed that G2B-10A cells promoted a ~3-fold increase in tumor growth, irrespective of PFA pre-treatment. These results indicate that soluble factors secreted by G2B-10A cells play a less important role in promoting R2-T1AS tumorigenesis in vivo, and that additional components are operative in the nude mouse xenograft assay. Finally, using array analysis, we found that both live and PFA-fixed G2B-10A cells induced R2-T1AS cells to secrete specific cytokines (IL-6 and GM-CSF), suggesting that cell-cell contact activates R2-T1AS cells.

Conclusions: Taken together, these data shift our understanding of adjacent benign epithelial cells in the cancer process, from passive, noncontributory cells to an active and tumor-promoting vicinal cell population that may have significant effects early, when benign cells outnumber malignant cells.

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Morphology and cellular composition of tumors harvested at day 7 and day 14. (A) Fluorescent sections of tumors harvested at 7 days post-inoculation (red fluorescence - R2-T1AS cells; green fluorescence - G2B-10A cells). Left panel: R2-T1AS - tumors resulting from the injection of R2-T1AS cells alone, partially composed of solid tissue (arrowhead) and partially of necrotic areas (asterisk). Right panel: R2-T1AS/G2B-10A - tumors resulting from the injection of R2-T1AS cells mixed with G2B-10A. Majority (8 of 10) of tumors at day 7 had cystic structure. The lumen of the cyst (L) was filled with fluid and cellular debris, and surrounded by G2B-10A (green fluorescence) and R2-T1AS (red fluorescence) cells. (B) Fluorescent sections of tumors harvested at 14 days post-inoculation (red fluorescence - R2-T1AS cells; green fluorescence - G2B-10A cells). Both R2-T1AS and R2-T1AS/G2B-10A mixed tumors were composed of solid tissue formed by R2-T1AS cells (red fluorescence). Scale bars: 1 mm.
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Figure 8: Morphology and cellular composition of tumors harvested at day 7 and day 14. (A) Fluorescent sections of tumors harvested at 7 days post-inoculation (red fluorescence - R2-T1AS cells; green fluorescence - G2B-10A cells). Left panel: R2-T1AS - tumors resulting from the injection of R2-T1AS cells alone, partially composed of solid tissue (arrowhead) and partially of necrotic areas (asterisk). Right panel: R2-T1AS/G2B-10A - tumors resulting from the injection of R2-T1AS cells mixed with G2B-10A. Majority (8 of 10) of tumors at day 7 had cystic structure. The lumen of the cyst (L) was filled with fluid and cellular debris, and surrounded by G2B-10A (green fluorescence) and R2-T1AS (red fluorescence) cells. (B) Fluorescent sections of tumors harvested at 14 days post-inoculation (red fluorescence - R2-T1AS cells; green fluorescence - G2B-10A cells). Both R2-T1AS and R2-T1AS/G2B-10A mixed tumors were composed of solid tissue formed by R2-T1AS cells (red fluorescence). Scale bars: 1 mm.

Mentions: To determine whether G2B-10A cells contribute to tumor masses, we harvested tumors from R2-T1AS/G2B-10A and R2-T1AS groups at 7 and 14 days post-inoculation and we also harvested tumors from all 3 groups (R2-T1AS/G2B-10A, R2-T1AS/G2B-10A(PFA) and R2-T1AS) at 28 days post-inoculation. Tumor sections were analyzed by fluorescence microscopy. As shown in Figure 8A, in the R2-T1AS-only group, the tumors 7 days post-injection were composed of solid tissue (arrowhead), and of necrotic areas of disaggregated cells and debris (asterisk). In the R2-T1AS/G2B-10A mixed group, 8 out of 10 tumors analyzed formed a cyst at 7 days, with the lumen filled with cellular debris and fluid, surrounded by green-marked G2B-10A cells and red-marked R2-T1AS cells. As shown in Figure 8B, at day 14 post-injection, the R2-T1AS/G2B-10A mixed tumors had filled the cyst and developed a solid structure, resulting in tumors larger than the R2-T1AS-only tumors. However, the benign G2B-10A cells were no longer detectable at 14 days. Similarly, at 28 days post-inoculation, tumors from all groups (R2-T1AS, R2-T1AS/G2B-10A and R2-T1AS/G2B-10A(PFA)) were composed solely of R2-T1AS cells forming solid masses, with tumors arising from R2-T1AS/G2B-10A injections and R2-T1AS/G2B-10A(PFA) injections showing no differences (see Additional file 1). This analysis reveals that G2B-10A benign cells disappeared from the tumor tissue within 2 weeks after inoculation. Nonetheless, the early and transient presence of G2B-10A (± PFA) cells resulted in higher tumorigenicity of R2-T1AS cells.


Benign mammary epithelial cells enhance the transformed phenotype of human breast cancer cells.

Poczobutt JM, Tentler J, Lu X, Schedin PJ, Gutierrez-Hartmann A - BMC Cancer (2010)

Morphology and cellular composition of tumors harvested at day 7 and day 14. (A) Fluorescent sections of tumors harvested at 7 days post-inoculation (red fluorescence - R2-T1AS cells; green fluorescence - G2B-10A cells). Left panel: R2-T1AS - tumors resulting from the injection of R2-T1AS cells alone, partially composed of solid tissue (arrowhead) and partially of necrotic areas (asterisk). Right panel: R2-T1AS/G2B-10A - tumors resulting from the injection of R2-T1AS cells mixed with G2B-10A. Majority (8 of 10) of tumors at day 7 had cystic structure. The lumen of the cyst (L) was filled with fluid and cellular debris, and surrounded by G2B-10A (green fluorescence) and R2-T1AS (red fluorescence) cells. (B) Fluorescent sections of tumors harvested at 14 days post-inoculation (red fluorescence - R2-T1AS cells; green fluorescence - G2B-10A cells). Both R2-T1AS and R2-T1AS/G2B-10A mixed tumors were composed of solid tissue formed by R2-T1AS cells (red fluorescence). Scale bars: 1 mm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 8: Morphology and cellular composition of tumors harvested at day 7 and day 14. (A) Fluorescent sections of tumors harvested at 7 days post-inoculation (red fluorescence - R2-T1AS cells; green fluorescence - G2B-10A cells). Left panel: R2-T1AS - tumors resulting from the injection of R2-T1AS cells alone, partially composed of solid tissue (arrowhead) and partially of necrotic areas (asterisk). Right panel: R2-T1AS/G2B-10A - tumors resulting from the injection of R2-T1AS cells mixed with G2B-10A. Majority (8 of 10) of tumors at day 7 had cystic structure. The lumen of the cyst (L) was filled with fluid and cellular debris, and surrounded by G2B-10A (green fluorescence) and R2-T1AS (red fluorescence) cells. (B) Fluorescent sections of tumors harvested at 14 days post-inoculation (red fluorescence - R2-T1AS cells; green fluorescence - G2B-10A cells). Both R2-T1AS and R2-T1AS/G2B-10A mixed tumors were composed of solid tissue formed by R2-T1AS cells (red fluorescence). Scale bars: 1 mm.
Mentions: To determine whether G2B-10A cells contribute to tumor masses, we harvested tumors from R2-T1AS/G2B-10A and R2-T1AS groups at 7 and 14 days post-inoculation and we also harvested tumors from all 3 groups (R2-T1AS/G2B-10A, R2-T1AS/G2B-10A(PFA) and R2-T1AS) at 28 days post-inoculation. Tumor sections were analyzed by fluorescence microscopy. As shown in Figure 8A, in the R2-T1AS-only group, the tumors 7 days post-injection were composed of solid tissue (arrowhead), and of necrotic areas of disaggregated cells and debris (asterisk). In the R2-T1AS/G2B-10A mixed group, 8 out of 10 tumors analyzed formed a cyst at 7 days, with the lumen filled with cellular debris and fluid, surrounded by green-marked G2B-10A cells and red-marked R2-T1AS cells. As shown in Figure 8B, at day 14 post-injection, the R2-T1AS/G2B-10A mixed tumors had filled the cyst and developed a solid structure, resulting in tumors larger than the R2-T1AS-only tumors. However, the benign G2B-10A cells were no longer detectable at 14 days. Similarly, at 28 days post-inoculation, tumors from all groups (R2-T1AS, R2-T1AS/G2B-10A and R2-T1AS/G2B-10A(PFA)) were composed solely of R2-T1AS cells forming solid masses, with tumors arising from R2-T1AS/G2B-10A injections and R2-T1AS/G2B-10A(PFA) injections showing no differences (see Additional file 1). This analysis reveals that G2B-10A benign cells disappeared from the tumor tissue within 2 weeks after inoculation. Nonetheless, the early and transient presence of G2B-10A (± PFA) cells resulted in higher tumorigenicity of R2-T1AS cells.

Bottom Line: Conditioned media derived from G2B-10A cells enhanced colony formation of R2-T1AS cells, whereas prior paraformaldehyde (PFA) fixation of G2B-10A cells abrogated this enhancement effect.These results indicate that soluble factors secreted by G2B-10A cells play a less important role in promoting R2-T1AS tumorigenesis in vivo, and that additional components are operative in the nude mouse xenograft assay.Finally, using array analysis, we found that both live and PFA-fixed G2B-10A cells induced R2-T1AS cells to secrete specific cytokines (IL-6 and GM-CSF), suggesting that cell-cell contact activates R2-T1AS cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biology Program, University of Colorado Denver, Aurora, CO 80045, USA.

ABSTRACT

Background: Recent research has yielded a wealth of data underscoring the key role of the cancer microenvironment, especially immune and stromal cells, in the progression of cancer and the development of metastases. However, the role of adjacent benign epithelial cells, which provide initial cell-cell contacts with cancer cells, in tumor progression has not been thoroughly examined. In this report we addressed the question whether benign MECs alter the transformed phenotype of human breast cancer cells.

Methods: We used both in vitro and in vivo co-cultivation approaches, whereby we mixed GFP-tagged MCF-10A cells (G2B-10A), as a model of benign mammary epithelial cells (MECs), and RFP-tagged MDA-MB-231-TIAS cells (R2-T1AS), as a model of breast cancer cells.

Results: The in vitro studies showed that G2B-10A cells increase the colony formation of R2-T1AS cells in both soft agar and clonogenicity assays. Conditioned media derived from G2B-10A cells enhanced colony formation of R2-T1AS cells, whereas prior paraformaldehyde (PFA) fixation of G2B-10A cells abrogated this enhancement effect. Moreover, two other models of benign MECs, MCF-12A and HuMECs, also enhanced R2-T1AS colony growth in soft agar and clonogenicity assays. These data reveal that factors secreted by benign MECs are responsible for the observed enhancement of the R2-T1AS transformed phenotype. To determine whether G2B-10A cells enhance the tumorigenic growth of co-injected R2-T1AS cells in vivo, we used the nude mouse xenograft assay. Co-injecting R2-T1AS cells with G2B-10A cells +/- PFA-fixation, revealed that G2B-10A cells promoted a ~3-fold increase in tumor growth, irrespective of PFA pre-treatment. These results indicate that soluble factors secreted by G2B-10A cells play a less important role in promoting R2-T1AS tumorigenesis in vivo, and that additional components are operative in the nude mouse xenograft assay. Finally, using array analysis, we found that both live and PFA-fixed G2B-10A cells induced R2-T1AS cells to secrete specific cytokines (IL-6 and GM-CSF), suggesting that cell-cell contact activates R2-T1AS cells.

Conclusions: Taken together, these data shift our understanding of adjacent benign epithelial cells in the cancer process, from passive, noncontributory cells to an active and tumor-promoting vicinal cell population that may have significant effects early, when benign cells outnumber malignant cells.

Show MeSH
Related in: MedlinePlus