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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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G2B-10A benign mammary epithelial cells increase the tumorigenicity of R2-T1AS cells in vivo independent of benign-cell secreted factors. Growth of xenograft tumors in nude mice resulting from the injection of 4 × 106 G2B-10A cells alone (G2B-10A, green line, n = 4), 1 × 106 R2-T1AS cells alone (R2-T1AS, red line, n = 10), 1 × 106 R2-T1AS cells mixed with 4 × 106 G2B-10A cells (R2-T1AS/G2B-10A, blue line, n = 6) and 1 × 106 R2-T1AS mixed with 4 × 106 PFA-fixed G2B-10A cells (R2-T1AS/G2B-10A(PFA), gray line, n = 10). Tumor size was measured every 7 days using a digital caliper and tumor volume was calculated using the formula 0.52 × length × width2. Data shown are derived from 2 independent experiments. *p < 0.05.
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Figure 7: G2B-10A benign mammary epithelial cells increase the tumorigenicity of R2-T1AS cells in vivo independent of benign-cell secreted factors. Growth of xenograft tumors in nude mice resulting from the injection of 4 × 106 G2B-10A cells alone (G2B-10A, green line, n = 4), 1 × 106 R2-T1AS cells alone (R2-T1AS, red line, n = 10), 1 × 106 R2-T1AS cells mixed with 4 × 106 G2B-10A cells (R2-T1AS/G2B-10A, blue line, n = 6) and 1 × 106 R2-T1AS mixed with 4 × 106 PFA-fixed G2B-10A cells (R2-T1AS/G2B-10A(PFA), gray line, n = 10). Tumor size was measured every 7 days using a digital caliper and tumor volume was calculated using the formula 0.52 × length × width2. Data shown are derived from 2 independent experiments. *p < 0.05.

Mentions: The studies included thus far address the role of benign mammary epithelial cells in enhancing the in vitro transformed phenotype of R2-T1AS breast cancer cells. To determine whether benign mammary epithelial cells also influence the phenotype of breast cancer cells in vivo, we examined the effects of G2B-10A cells on the tumorigenicity of R2-T1AS cells in a nude mouse xenograft model. First, we injected 4 × 106 G2B-10A cells alone to verify that these cells were not tumorigenic, and indeed, G2B-10A cells did not form any tumors (Figure 7, green line). To establish the ability of R2-T1AS cells to form tumors in mice, we injected 1 × 106 R2-T1AS cells alone, which formed tumors that on average measured 545 mm3 in volume on day 28 after inoculation (Figure 7, red line). To determine whether G2B-10A cells influenced the tumorigenicity of R2-T1AS breast cancer cells, we co-injected 4 × 106 G2B-10A cells with1 × 106 R2-T1AS cells, maintaining the increased ratio of benign to malignant cells, as used in the in vitro studies. As expected from in vitro results, the volume of these R2-T1AS/G2B-10A mixed tumors reached 1521 mm3 on day 28, which is about 3-fold larger than R2-T1AS-only tumors (Figure 7, blue line). The data described here are derived from 2 separate experiments. We also performed 3 additional separate experiments (not shown), which yielded similar results. The result shown in Figure 7 recapitulated the in vitro studies, in which the G2B-10A cells enhanced the transformed phenotype of R2-T1AS. Next, we tested the hypothesis that the enhanced tumorigenicity of R2-T1AS in vivo is mediated by soluble factors secreted by G2B-10A cells, as the in vitro data suggest (Figure 3). To this end, we co-injected 1 × 106 R2-T1AS cells with 4 × 106 paraformaldehyde-fixed G2B-10A(PFA) cells. Unexpectedly, fixation of G2B-10A cells with PFA failed to reduce the promotion of tumor growth, as the mixed R2-T1AS/G2B-10A(PFA) grafts reached 1813 mm3, which is equivalent to the R2-T1AS/G2B-10A mixed tumors. While this result is not fully consistent with what we observed in vitro, the complexity of the in vivo intact mammary gland may require several influences (secreted factors, direct cell contact, stromal contributions, etc) to promote optimal growth of R2-T1AS tumors in the nude mouse xenograft model.


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)

G2B-10A benign mammary epithelial cells increase the tumorigenicity of R2-T1AS cells in vivo independent of benign-cell secreted factors. Growth of xenograft tumors in nude mice resulting from the injection of 4 × 106 G2B-10A cells alone (G2B-10A, green line, n = 4), 1 × 106 R2-T1AS cells alone (R2-T1AS, red line, n = 10), 1 × 106 R2-T1AS cells mixed with 4 × 106 G2B-10A cells (R2-T1AS/G2B-10A, blue line, n = 6) and 1 × 106 R2-T1AS mixed with 4 × 106 PFA-fixed G2B-10A cells (R2-T1AS/G2B-10A(PFA), gray line, n = 10). Tumor size was measured every 7 days using a digital caliper and tumor volume was calculated using the formula 0.52 × length × width2. Data shown are derived from 2 independent experiments. *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 7: G2B-10A benign mammary epithelial cells increase the tumorigenicity of R2-T1AS cells in vivo independent of benign-cell secreted factors. Growth of xenograft tumors in nude mice resulting from the injection of 4 × 106 G2B-10A cells alone (G2B-10A, green line, n = 4), 1 × 106 R2-T1AS cells alone (R2-T1AS, red line, n = 10), 1 × 106 R2-T1AS cells mixed with 4 × 106 G2B-10A cells (R2-T1AS/G2B-10A, blue line, n = 6) and 1 × 106 R2-T1AS mixed with 4 × 106 PFA-fixed G2B-10A cells (R2-T1AS/G2B-10A(PFA), gray line, n = 10). Tumor size was measured every 7 days using a digital caliper and tumor volume was calculated using the formula 0.52 × length × width2. Data shown are derived from 2 independent experiments. *p < 0.05.
Mentions: The studies included thus far address the role of benign mammary epithelial cells in enhancing the in vitro transformed phenotype of R2-T1AS breast cancer cells. To determine whether benign mammary epithelial cells also influence the phenotype of breast cancer cells in vivo, we examined the effects of G2B-10A cells on the tumorigenicity of R2-T1AS cells in a nude mouse xenograft model. First, we injected 4 × 106 G2B-10A cells alone to verify that these cells were not tumorigenic, and indeed, G2B-10A cells did not form any tumors (Figure 7, green line). To establish the ability of R2-T1AS cells to form tumors in mice, we injected 1 × 106 R2-T1AS cells alone, which formed tumors that on average measured 545 mm3 in volume on day 28 after inoculation (Figure 7, red line). To determine whether G2B-10A cells influenced the tumorigenicity of R2-T1AS breast cancer cells, we co-injected 4 × 106 G2B-10A cells with1 × 106 R2-T1AS cells, maintaining the increased ratio of benign to malignant cells, as used in the in vitro studies. As expected from in vitro results, the volume of these R2-T1AS/G2B-10A mixed tumors reached 1521 mm3 on day 28, which is about 3-fold larger than R2-T1AS-only tumors (Figure 7, blue line). The data described here are derived from 2 separate experiments. We also performed 3 additional separate experiments (not shown), which yielded similar results. The result shown in Figure 7 recapitulated the in vitro studies, in which the G2B-10A cells enhanced the transformed phenotype of R2-T1AS. Next, we tested the hypothesis that the enhanced tumorigenicity of R2-T1AS in vivo is mediated by soluble factors secreted by G2B-10A cells, as the in vitro data suggest (Figure 3). To this end, we co-injected 1 × 106 R2-T1AS cells with 4 × 106 paraformaldehyde-fixed G2B-10A(PFA) cells. Unexpectedly, fixation of G2B-10A cells with PFA failed to reduce the promotion of tumor growth, as the mixed R2-T1AS/G2B-10A(PFA) grafts reached 1813 mm3, which is equivalent to the R2-T1AS/G2B-10A mixed tumors. While this result is not fully consistent with what we observed in vitro, the complexity of the in vivo intact mammary gland may require several influences (secreted factors, direct cell contact, stromal contributions, etc) to promote optimal growth of R2-T1AS tumors in the nude mouse xenograft model.

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