Local application of bacteria improves safety of Salmonella -mediated tumor therapy and retains advantages of systemic infection

Abstract

Cancer is a devastating disease and a large socio-economic burden. Novel therapeutic solutions are on the rise, although a cure remains elusive. Application of microorganisms represents an ancient therapeutic strategy, lately revoked and refined via simultaneous attenuation and amelioration of pathogenic properties. Salmonella Typhimurium has prevailed in preclinical development. Yet, using virulent strains for systemic treatment might cause severe side effects. In the present study, we highlight a modified strain based on Salmonella Typhimurium UK-1 expressing hexa-acylated Lipid A. We corroborate improved anti-tumor properties of this strain and investigate to which extent an intra-tumoral (i.t.) route of infection could help improve safety and retain advantages of systemic intravenous (i.v.) application. Our results show that i.t. infection exhibits therapeutic efficacy against CT26 and F1.A11 tumors similar to a systemic route of inoculation. Moreover, i.t. application allows extensive dose titration without compromising tumor colonization. Adverse colonization of healthy organs was generally reduced via i.t. infection and accompanied by less body weight loss of the murine host. Despite local application, adjuvanticity remained, and a CT26-specific CD8+ T cell response was effectively stimulated. Most interestingly, also secondary tumors could be targeted with this strategy, thereby extending the unique tumor targeting ability of Salmonella. The i.t. route of inoculation may reap the benefits of systemic infection and aid in safety assurance while directing potency of an oncolytic vector to where it is most needed, namely the primary tumor.

Keywords: E. coli; Intra-tumoral injection; Salmonella; bacteria mediated tumor therapy; murine tumor model.

Figure 1. Tumor development upon intravenous and intra-tumoral infection with Salmonella and probiotic E. coli

CT26 tumor-bearing mice were infected with 5×10⁶ CFU SF200 (ΔlpxR9 ΔpagL7 ΔpagP8 ΔaroA ΔydiV ΔfliF) (A), Symbioflor-2 (C) and E. coli Nissle (D). Considering more resilient tumors, F1.A11 tumor-bearing mice were infected with 5×10⁶ SF200 (B). Straight lines depict i.v. infection and dotted lines i.t. infection. Tumor volumes were calculated on the basis of caliper measurements. PBS served as a negative control. Displayed are values of mean ± SEM. Results are representative of two independent experiments with five replicates in each group.

Figure 2. Intra-tumoral inoculation of Salmonella causes manifestations in the CT26 tumor similar to systemic infection

CT26 tumor-bearing mice were infected with 5×10⁶ CFU SF200 (ΔlpxR9 ΔpagL7 ΔpagP8 ΔaroA ΔydiV ΔfliF) via i.t. and i.v. routes of inoculation (top and bottom row, resp.). 48 hpi, tumors were isolated, embedded in paraffin and prepared for immune histochemical staining. Similar histological profiles between i.t. and i.v. infections: similar degree of necrosis formation and hypoxia, dispersion of salmonellae in and beyond necrotic center, and presence of neutrophils in immediate proximity to the salmonellae. Images displayed are representative of four replicates. “N” denotes areas of necrosis. Hypoxia was stained with antibodies against metabolites of pimonidazole-HCl administered i.v. 30 mins prior to isolation. Myeloperoxidase (MPO) denotes presence of neutrophilic granulocytes, and salmonellae was stained using a specific antibody. Differential staining was performed on consecutive sections. Scale bar corresponds to 100 μm. Images representative of at least 3 replicates are displayed.

Figure 3. Safety evaluation upon intravenous and intra-tumoral infection with Salmonella and probiotic E. coli

CT26 tumor bearing mice were infected i.v. and i.t. with 5×10⁶ CFU SF200 (ΔlpxR9 ΔpagL7 ΔpagP8 ΔaroA ΔydiV ΔfliF) and probiotic E. coli. (A-C) Blood, spleen, liver and tumor were analyzed for bacterial burden by plating serial dilutions of tissue homogenates. CFU counts were determined 48 hpi. Significantly lower numbers were observed during i.t. infections. (D-F) Body weight as indicator for the general health status of mice. Again, i.t. infection resulted in reduced body weight loss. PBS served as negative control. Displayed are medians with range. Results are representative of two independent experiments with five replicates per group. *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 4. Intra-tumoral infection with Salmonella promotes higher tumor specificity and faster tumor colonization even at lower doses

CT26- tumor bearing mice were infected i.v. and i.t. with a dose of 5×10⁶ and 5×10⁵ Salmonella variant SF201 (ΔlpxR9 ΔpagL7 ΔpagP8 ΔaroA ΔssrA::Km). Tumors (A), blood (B), spleen (C) and liver (D). Bacterial burden was determined by plating serial dilutions of tissue homogenates. CFU were analyzed 36 hpi. (E) Tumor volumes were calculated on the basis of caliper measurements following infection with SF201. PBS served as negative control. (F) Body weight measurement served as indicator for the general health status. I.t. infection resulted in a significantly reduced bacterial burden and body weight decrease. PBS served as negative control. Displayed are medians with range. Results are representative of two independent experiments with five replicates per group.

Figure 5. Intra-tumoral infection induces innate and adaptive immune responses

(A) TNF-α levels in sera of CT26 tumor bearing mice isolated 1.5 h after infection with SF200 (ΔlpxR9 ΔpagL7 ΔpagP8 ΔaroA ΔydiV ΔfliF). (B) CT26 tumor development in Rag1^−/− mice reconstituted with CD8⁺ T cells at the time of CT26 inoculation. 3×10⁶ CD8⁺ T cells were adoptively transferred from uninfected CT26 bearing mice (“CD8⁺ T cells (CT26)”) or CT26 tumor bearing mice treated with SF200 ((“CD8⁺ T cells (Infection)”). PBS served as negative control. (C) Endpoint tumor volume at day 14 post transfer. Displayed are values of mean ± SEM. Results are representative of two independent experiments with five replicates per group. *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 6. Intra-tumoral infection allows effective colonization of secondary CT26 tumors

(A) Mice bearing two CT26 tumors were infected i.v. or i.t. into one tumor with 5×10⁶ CFU SF202 (ΔlpxR9 ΔpagL7 ΔpagP8 ΔaroA ΔydiV ΔfliF pHL304). Anesthetized mice were analyzed at 0, 1, 2 and 3 dpi using a non-invasive in vivo imaging system (IVIS200). (B) Bacterial colonization of tumors was determined by plating serial dilutions of tissue homogenates. CFU were analyzed 12, 24, 48 and 72 hpi to match imaging time points. For i.t. infection, primary and secondary tumors are denoted 1° and 2°, respectively. Displayed are medians with range. Results are representative of two independent experiments with five replicates in each group.