Extended Data Fig. 9: Mal-LbL NP treatment primes the TME for CPI treatment.

a-d B6C3F1 mice (n = 5 animals/group) inoculated with 10⁶ HM-1 tumor cells on day 0 were treated on day 10 with 20 µg of IL-12 as a free cytokine or conjugated to Mal-LbL NPs. Two days after dosing ascites and i.p. tumor nodules (primarily omentum tissue) were harvested and processed for flow cytometry analysis. Shown are quantitation of the percentage of T regs among T cells in ascites (a), PD1 positive CD4 T cell (b) and CD8 T cell (c) in ascites, CD25⁺FoxP3⁻ of CD4 T cells (d) in ascites, CD25⁺of CD8 T cells (e) in ascites, T regs of T cells in tumor (f), CD25⁺FoxP3⁻ of CD4 T cells in tumor (g), PD1⁺TIM3⁻ of CD4 T cells in tumors (h), PD1⁺TIM3⁺ of CD4 T cells in tumors (i), PD1⁺TIM3⁻ of CD8 T cells in tumors (j), PD1⁺TIM3⁺ of CD8 T cells in tumors (k), and CD25⁺ of CD8 T cells (l). m, B6C3F1 mice (n = 10 animals/group) inoculated with 10⁶ HM-1-luc tumor cells on day 0 were treated on days 7 and 14 with 20 µg of IL-12 as a free cytokine or conjugated to Mal-LbL Mice were also treated with 250 µg of anti-PD1 and 100 µg of anti-CTLA4 i.p. on days 8 and 15. Shown are the relative body weight of mice (black arrows indicate IL-12 treatment and blue indicate CPI). None of the treated mice showed significant drop in bodyweight compared to dextrose control. n-p, B6C3F1 mice (2 cohorts with n = 5 animals/group/cohort) inoculated with 10⁶ HM-1-luc tumor cells on day 0 were treated on days 7 and 14 with 20 µg of IL-12 as a free cytokine or conjugated to Mal-LbL or Ni-LbL. Mice were also treated with 250 µg of anti-PD1 and 100 µg of anti-CTLA4 i.p. on days 8 and 15. Shown are the in vivo IVIS whole-animal i.p. BLI readings (mean ± s.d., n). On day 150, surviving mice were rechallenged with either 3×10⁵ (o) or 10⁶ (p) HM-1-luc tumor cells i.p. Shown are the in vivo IVIS whole-animal i.p. BLI readings post rechallenge (mean ± s.d). In all panels, each point corresponds to a single animal, with values shown as mean ± standard deviation.

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