Fig. 4. Vancomycin blunts CTX-induced pTh17 differentiation which is mandatory for the tumoricidal activity of chemotherapy.
(A). After a 3 week-long pretreatment with broad-spectrum ATB, DBA2 mice were inoculated with P815 mastocytomas (day 0), treated at day 6 with CTX (arrow) and tumor growth was monitored. Tumor growth kinetics are shown in Fig. S9A and percentages of tumor-free mice at sacrifice are depicted for two experiments of 11–14 mice/group. (B). MCA205 sarcoma were inoculated at day 0 in specific pathogen-free (SPF) or germ-free (GF) mice that were optionally mono-associated with segmented filamentous bacteria (SFB), treated with CTX (arrow) and monitored for growth kinetics (means±SEM). One representative experiment (n=5–8 mice/group) out of two to three is shown for GF mice and two pooled experiments (n=14 mice/group) for SPF mice (C). After a 3 week- conditioning with vancomycin or colistin, C57BL/6 mice were inoculated with MCA205 sarcomas (day 0), treated at day 12–15 with CTX (arrow) and tumor growth was monitored. Concatenated data (n=15–20 mice/group) from two independent experiments are shown for colistin treatment and one representative experiment (n=6 mice/group) for vancomycin treatment. (D). Eight week-old KP (KrasLSL-G12D/WT; p53Flox/Flox) mice received an adenovirus expressing the Cre recombinase (AdCre) by intranasal instillation to initiate lung adenocarcinoma (d0). Vancomycin was started for a subgroup of mice (“Chemo + Vanco”) on d77 post-AdCre. One week after the start of vancomycin, CTX-based chemotherapy was applied i.p. to mice that only received chemotherapy (“Chemo”) or those that received in parallel vancomycin (“Chemo + Vanco”). Mice received chemotherapy on d84, d91 and d98. A control group was left untreated ("Co"). Data show the evolution of total lung tumor volumes (mean±SEM) assessed by non invasive imaging between d73 and d100 in 6–12 mice/group. (E). As in Fig. 3C, we determined the number of pTh17 cells in spleens from untreated or vancomycin treated mice bearing established (15–17 days) MCA205 tumors, 7 days after CTX treatment. Each dot represents one mouse from 2 pooled experiments. (F). Flow cytometric analyses of CD3+ and CD4+IFNγ+ T cells were performed by gating on CD45+ live tumor-infiltrating lymphocytes (TILs) extracted from day 18 established MCA205 tumors (8 days post-CTX) in water or vancomycin-treated mice. Each dot representing one mouse from up to four pooled experiments. (G). MCA205 tumors established in WT mice pretreated for 3 weeks with water or vancomycin were injected with CTX (arrow), and tumor growth was monitored. At day 7 post-CTX, 3 million of ex vivo generated Th17 or pTh17 CD4+ T cells were injected intravenously. Up to three experiments comprising 2–10 mice/group were pooled. Data were either analyzed with the t-test, linear model or generalized linear model. *, p<0.5, **, p<0.1, ***, p<0.001, ns, non significant.