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Research article
Connie Rogers
Pennsylvania State University University Park : Penn State
Corresponding Author
ORCiD: https://orcid.org/0000-0003-4072-2721
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: The murine 4T1.2 triple-negative breast cancer model is widely used, but is poorly immunogenic with no defined tumor-associated antigens. A modified 4T1.2 model has been developed that stably expresses a surrogate tumor antigen, human epidermal growth factor receptor-2 (HER2). The goal of the current study was to characterize host immune responses in the 4T1.2-HER2 tumor model, focusing on the tumor microenvironment (TME) during the early stage of tumor development.
Methods: Female BALB/c mice were orthotopically inoculated with 4T1.2-HER2 tumor cells and sacrificed at day (D) 6, 9, 12, 15 and 18 post tumor inoculation. The phenotype and function of tumor-infiltrating immune cells were assessed.
Results: 4T1.2 and 4T1.2-HER2 tumor cells had similar proliferation rates in vitro. In contrast to the rapid progression of the parental 4T1.2 model, the 4T1.2-HER2 model demonstrated initial tumor growth followed by spontaneous tumor regression by D18 post tumor inoculation, which was not observed in scid mice. Following tumor regression, mice demonstrated either a second phase of tumor outgrowth or complete tumor rejection. Within the TME, the percentage of T cells was reduced at D9 and increased during tumor regression through D18 (p<0.05), whereas the percentage of myeloid-derived suppressor cells (MDSCs) increased during the initial tumor growth and was reduced by D18 (p<0.01). There was a stepwise increase in the percentage of IFNg+, IL-2+ and perforin+ T cells and NK cells peaking at D12-15. Furthermore, tumor regression occurred concurrently with HER2-specific IFNg production from tumor-infiltrating immune cells at D12 and D15 (p<0.05). During the second phase of 4T1.2-HER2 tumor growth, tumor volume was negatively correlated with immune infiltration (r=0.662, p=0.052).
Conclusions: These results suggest that the integration of a surrogate tumor antigen, human HER2, into the clinically relevant, yet poorly immunogenic 4T1.2 breast cancer model enhanced its immunogenicity and induced HER2-specific immune responses.
Keywords
breast cancer, immunogenicity, HER2 antigen, antigen-specific immune response, TILs
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This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfile1.pdf
Additional file 1: Table S1. List of antibodies used for flow cytometric analyses of tumor cell lines. Table S2. List of antibodies used for flow cytometric analyses of immune cells. Table S3. Temporal changes in splenic immune cells expressing activation and memory markers during 4T1.2-HER2 initial tumor growth and regression. Table S4. Temporal changes in IFNƔ secretion (pg/mL) by splenic immune cells in response to different stimuli during 4T1.2-HER2 initial tumor growth and regression.
- Additionalfile2.pdf
Figure S1. Expression of MHC and costimulatory molecules in vitro was not different between 4T1.2 and 4T1.2-HER2 tumor cell lines. Figure S2. 4T1.2-HER2 initial tumor growth and regression was associated with changes in splenic effector and immunosuppressive cells. Figure S3. 4T1.2-HER2 initial tumor growth and regression was associated with changes in splenic myeloid cells and Tregs. Figure S4. 4T1.2-HER2 initial tumor growth and regression was associated with changes in tumor-infiltrating myeloid cells and Tregs. Figure S5. 4T1.2-HER2 initial tumor growth and regression was associated with changes in splenic immune cells expressing effector molecules. Figure S6. 4T1.2-HER2 tumor regression occurred concurrently with HER2-specific IFNγ production by splenic immune cells. Figure S7. Tumor volume during the second phase of 4T1.2-HER2 tumor growth was not correlated with the percentage of tumor-infiltrating effector T cells or Tregs. Figure S8. Tumor volume during the second phase of 4T1.2-HER2 tumor growth or tumor rejection was correlated with the percentage of splenic effector cells and MDSCs. Figure S9. Splenic effector to immunosuppressive cell ratios were higher in mice that rejected the tumor compared to mice that demonstrated the second phase of tumor growth.
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This is a preprint. It has not completed peer review.
Research article
Connie Rogers
Pennsylvania State University University Park : Penn State
Corresponding Author
ORCiD: https://orcid.org/0000-0003-4072-2721
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 09 Feb, 2021
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: The murine 4T1.2 triple-negative breast cancer model is widely used, but is poorly immunogenic with no defined tumor-associated antigens. A modified 4T1.2 model has been developed that stably expresses a surrogate tumor antigen, human epidermal growth factor receptor-2 (HER2). The goal of the current study was to characterize host immune responses in the 4T1.2-HER2 tumor model, focusing on the tumor microenvironment (TME) during the early stage of tumor development.
Methods: Female BALB/c mice were orthotopically inoculated with 4T1.2-HER2 tumor cells and sacrificed at day (D) 6, 9, 12, 15 and 18 post tumor inoculation. The phenotype and function of tumor-infiltrating immune cells were assessed.
Results: 4T1.2 and 4T1.2-HER2 tumor cells had similar proliferation rates in vitro. In contrast to the rapid progression of the parental 4T1.2 model, the 4T1.2-HER2 model demonstrated initial tumor growth followed by spontaneous tumor regression by D18 post tumor inoculation, which was not observed in scid mice. Following tumor regression, mice demonstrated either a second phase of tumor outgrowth or complete tumor rejection. Within the TME, the percentage of T cells was reduced at D9 and increased during tumor regression through D18 (p<0.05), whereas the percentage of myeloid-derived suppressor cells (MDSCs) increased during the initial tumor growth and was reduced by D18 (p<0.01). There was a stepwise increase in the percentage of IFNg+, IL-2+ and perforin+ T cells and NK cells peaking at D12-15. Furthermore, tumor regression occurred concurrently with HER2-specific IFNg production from tumor-infiltrating immune cells at D12 and D15 (p<0.05). During the second phase of 4T1.2-HER2 tumor growth, tumor volume was negatively correlated with immune infiltration (r=0.662, p=0.052).
Conclusions: These results suggest that the integration of a surrogate tumor antigen, human HER2, into the clinically relevant, yet poorly immunogenic 4T1.2 breast cancer model enhanced its immunogenicity and induced HER2-specific immune responses.
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