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. 2021 Nov 16;10(1):108–125. doi: 10.1158/2326-6066.CIR-21-0454

Figure 4.

Figure 4. Intratumoral HER3-DC1 administration elicits peptide-specific immune responses and delays tumor growth. A, Tumor growth in the 4T1 murine mammary carcinoma model. BALB/c mice bearing subcutaneous 4T1 tumors received either intratumoral PBS (black), unpulsed mature DC1 (blue), or HER3 peptide–pulsed DC1 (red; n = 10 mice/group), starting on day 7 when tumors were palpable. Tumor growth was monitored until endpoint and was compared between control and HER3-DC1, as well as between unpulsed DC1 and HER3-DC1. *, control versus HER3-DC1; #, unpulsed DC1 versus HER3-DC1. B, Individual tumor growth for each mouse from control (black)-, unpulsed DC1 (blue)–, and HER3-DC1 (red)–treated groups. C, Percent survival in the 4T1 mouse model. Control, black; unpulsed DC1, blue; HER3-DC1, red. D, Intratumoral CD3+CD4+ and CD3+CD8+ T-cell infiltration per milligram of tumor in control (black)-, unpulsed DC1 (blue)–, and HER3-DC1 (red)–treated mice. Absolute number of immune cells was compared between control and HER3-DC1 groups. E, Frequency of CD62L+CD44+ central memory (CM), CD62L−CD44+ effector memory (EM), and CD62L−CD44− effector (Eff) T-cell populations within intratumoral CD4+ cells between control- (black) and HER3-DC1–treated (red) tumors. The unpulsed DC1 (blue) group was not included in any statistical analyses. F, Absolute number of CD3+CD4+ and CD3+CD8+ T cells in lymph nodes of control (black)-, intratumoral unpulsed DC1 (blue)–, and HER3-DC1 (red)–treated mice. Cell numbers were compared in control versus HER3-DC1 groups. Data shown are the representative from three independent experiments. G, Absolute numbers of CD4+ CM, EM, and Eff T-cell populations in lymph nodes of control (black), unpulsed DC1 (blue), and HER3-DC1 (red) mice. Data shown are the representative from three independent experiments. H, Lymphocytes from the lymph nodes of control and treated mice were cocultured with DC1 pulsed with individual HER3 or OT-II (negative control) peptides. Culture supernatants were collected after 72 hours, and IFN-γ was measured by ELISA (control: black bar; HER3-DC1: red bar). I, Total protein isolated from in vivo tumor samples was analyzed by Western blotting to compare HER3 protein expression after intratumoral HER3-DC1 (green) administration with respect to the control (black). β-Actin: loading control. Data represented as mean ± SEM with statistical significance determined using multiple t test without correction for multiple comparisons. Each row was analyzed individually, without assuming a consistent SD. A log-rank (Mantel–Cox) test was used to determine differences between the survival curves. Unpaired two-tailed t test was performed to analyze Western blot data. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; #, P ≤ 0.01.

Intratumoral HER3-DC1 administration elicits peptide-specific immune responses and delays tumor growth. A, Tumor growth in the 4T1 murine mammary carcinoma model. BALB/c mice bearing subcutaneous 4T1 tumors received either intratumoral PBS (black), unpulsed mature DC1 (blue), or HER3 peptide–pulsed DC1 (red; n = 10 mice/group), starting on day 7 when tumors were palpable. Tumor growth was monitored until endpoint and was compared between control and HER3-DC1, as well as between unpulsed DC1 and HER3-DC1. *, control versus HER3-DC1; #, unpulsed DC1 versus HER3-DC1. B, Individual tumor growth for each mouse from control (black)-, unpulsed DC1 (blue)–, and HER3-DC1 (red)–treated groups. C, Percent survival in the 4T1 mouse model. Control, black; unpulsed DC1, blue; HER3-DC1, red. D, Intratumoral CD3+CD4+ and CD3+CD8+ T-cell infiltration per milligram of tumor in control (black)-, unpulsed DC1 (blue)–, and HER3-DC1 (red)–treated mice. Absolute number of immune cells was compared between control and HER3-DC1 groups. E, Frequency of CD62L+CD44+ central memory (CM), CD62L−CD44+ effector memory (EM), and CD62L−CD44− effector (Eff) T-cell populations within intratumoral CD4+ cells between control- (black) and HER3-DC1–treated (red) tumors. The unpulsed DC1 (blue) group was not included in any statistical analyses. F, Absolute number of CD3+CD4+ and CD3+CD8+ T cells in lymph nodes of control (black)-, intratumoral unpulsed DC1 (blue)–, and HER3-DC1 (red)–treated mice. Cell numbers were compared in control versus HER3-DC1 groups. Data shown are the representative from three independent experiments. G, Absolute numbers of CD4+ CM, EM, and Eff T-cell populations in lymph nodes of control (black), unpulsed DC1 (blue), and HER3-DC1 (red) mice. Data shown are the representative from three independent experiments. H, Lymphocytes from the lymph nodes of control and treated mice were cocultured with DC1 pulsed with individual HER3 or OT-II (negative control) peptides. Culture supernatants were collected after 72 hours, and IFN-γ was measured by ELISA (control: black bar; HER3-DC1: red bar). I, Total protein isolated from in vivo tumor samples was analyzed by Western blotting to compare HER3 protein expression after intratumoral HER3-DC1 (green) administration with respect to the control (black). β-Actin: loading control. Data represented as mean ± SEM with statistical significance determined using multiple t test without correction for multiple comparisons. Each row was analyzed individually, without assuming a consistent SD. A log-rank (Mantel–Cox) test was used to determine differences between the survival curves. Unpaired two-tailed t test was performed to analyze Western blot data. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; #, P ≤ 0.01.