Fig. 4: Intratumoral CarboCell TLR:TGFb induces systemic anticancer immune activity and immunological memory.

a Treatment schedule (CT26 tumors, CC Supplementary Table 1). Mice bearing two established CT26 tumors were injected intratumorally in one tumor with CarboCell. b Individual tumor growth curves and (c) survival plots of mice bearing two CT26 tumors [start size ~100 mm³ (injected tumor), start size ~87 mm³ (uninjected tumor), n = 9 (UT) and n = 15 (CarboCell TLR:TGFb)]. d Flow cytometry analysis (one-way ANOVA with Tukey post-test) of %CD36 (of Tregs) (F(2,19) = 20.99, P < 0.0001) and %Tregs (of CD45 + ) (F(2,19) = 0.6498, P = 0.5334) in tumors 3 days [n = 6 (UT)] or 14 days [n = 5 (CarboCell TLR:TGFb)] after injection of CarboCell. Mice bearing two established CT26 tumors were injected intratumorally in one tumor with CarboCell TLR:TGFb (7.5 mg/kg:20 mg/kg, CC (Supplementary Table 1)). Untreated mice were included as controls. For gating strategy see Supplementary Fig. 18. e TCRβ CDR3 nucleotide sequence overlap (two-tailed unpaired t-test) between intratumoral TCRβ CarboCell injected tumors and uninjected tumors from mouse bilateral tumor model [n = 6 (UT), n = 4 (CarboCell TLR:TGFb)]. f Morisita overlap index of intratumor TCRβ in CarboCell injected (R) and contralateral uninjected tumor (L) from CarboCell CarboCell TLR:TGFb treated mice [n = 4 (M01-M04)]. g, h Lung metastases were evaluated in mice bearing s.c. 4T1 flank tumors after three weekly intratumoral injections of CarboCell TLR (7.5 mg/kg), CarboCell TGFb (20 mg/kg), or CarboCell TLR:TGFb (7.5 mg/kg:20 mg/kg) starting at day 7 post inoculation (CC (Supplementary Table 1)), or no treatment (UT) [n = 8 (CarboCell groups), n = 7 (UT)]. 24 days post inoculation, lung metastases were quantified by 6-thioguanine clonogenic assay (Supplementary Fig. 19). g Percent of mice displaying complete clearance of pulmonary metastases. h Analysis of lung colonies (one-way ANOVA with Tukey post-test) (F(3,27) = 5.765, P = 0.0035) i Flow cytometry evaluation of tumor arginase-1 expression (MFI) in 4T1 tumors injected with CarboCell TLR:TGFb (7.5 mg/kg:20 mg/kg, CC (Supplementary Table 1)) analyzed (one-way ANOVA with Tukey post-test) one (1d), three (3d), and seven days (7d) after treatment [n = 6 (UT), n = 5 (d1) n = 5 (d3) n = 6 (d7)] on Mo-MDSCs (F(3,18) = 8.852, P = 0.0008), PMos (F(3,18) = 22.24, P < 0.0001), and TAMs (F(3,18) = 7.347, P = 0.0020). For gating strategy see Supplementary Fig. 20. j Treatment schedule (CT26 tumors, splenocyte transfer). Mice bearing established CT26 tumors were treated with intraperitoneal injection (i.p.) of cyclophosphamide (CTX) one day prior to splenocyte transfer. The mice received either no splenocytes, splenocytes from naïve mice, splenocytes from mice bearing established CT26 tumors, or splenocytes from mice rejecting two CT26 tumors (data shown in a–c). k Mean tumor growth curves and (l) survival plots of mice bearing CT26 tumors [palpable tumors, n = 8/group]. The results are presented as mean ± SEM. Source data and exact P values are provided as a Source Data file. Illustrations created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.