Biotech

Remolding the tumor microenvironment by bacteria augments adoptive T cell therapy in advanced-stage solid tumors

Time Stamp: November 21, 2024 7:00 PM
Source Node: 751612

Intratumor E. coli modulates tumor physical and physiochemical microenvironment

We first determined the cytokine and chemokine profile in B16F10-OVA tumors following intratumor (i.t.) injection of E. coli. Luminex assay showed an elevation in TNF-α and nearly all chemokines, with special notice to CCL3, CCL4, CCL5, CCL19, CXCL10, and CXCL11, which were reported as crucial CD8 T cell recruiting chemokines (Fig. 1b and Supplementary Fig. 1). We observed 19.4-fold, 38.1-fold, 32.4-fold, 1.89-fold, 7.44-fold, and 2.36-fold increments of CCL3, CCL4, CCL5, CCL19, CXCL10, and CXCL11, respectively, in the E. coli-treated group. We then explored the impact of E. coli on the physical levels of tumor tissues. Tumor vasculatures have been reported to inhibit T cell extravasation, suppress T cell activities, and mediate FasL-induced T cell apoptosis.³¹ The normalization of tumor vessels could assist in the infiltration of cytotoxic T cells. We stained tumor vasculatures with CD31 and observed intratumor E. coli could destroy tumor vasculatures at the injection site and augment the formation and normalization of tumor vasculature in the periphery, which could facilitate the extravasation of circulating T cells into tumor tissues (Fig. 1c and Supplementary Fig. 2).^30,32

Intratumor E. coli treatment augments OT-I T cell infiltration into solid tumors

As E. coli elevated intratumor T cell-centered chemokines, we preliminarily tested whether the tumor interstitial fluid could directly promote T cell infiltration. We derived OT-I T cells as a model for tumor-reactive T cells and assessed the CD8 T cell population along with their tumor-killing efficacy (Supplementary Fig. 3). We placed B16F10-OVA cells and tumor fluid content at the bottom well with the upper well loaded with CFSE-labeled OT-I T cells. The flowcytometric analysis showed a higher OT-I T cells to beads ratio in the E. coli group, ~3.95 folds of that in the PBS group (Fig. 2a). In vivo imaging system (IVIS) image also revealed a 0.52-fold elevation of CFSE intensity in the E. coli group (Supplementary Fig. 4). To further validate the T cell recruiting efficacy of the elevated intratumor chemokine profiles induced by E. coli, we tested on whole tumor tissue. The resected whole B16F10-OVA tumor tissues with pretreatment of i.t. PBS or i.t. E. coli were co-cultured with OT-I T cells. IVIS image determined a higher Cy5 signal of OT-I T cells (~1.87 folds of the control group) in the E. coli group (Fig. 2b). To ascertain whether intratumor E. coli treatment could directly promote OT-I T cell infiltration into the tumor tissues, we repeated the co-culture assay as Fig. 2b, with whole tumor tissues substituted by surgical-derived half tumors. In comparison with the whole tumor setting, the inner tissue of the half tumors could directly interface with OT-I T cells. As a result, a higher Cy5.5 signal of OT-I T cells was revealed in the E. coli group, about 1.21-fold of that in the control group (Supplementary Fig. 5).

Based on the above in vitro evidence, we then determined the tumor infiltration condition of OT-I T cells in vivo. OT-I T cells were pre-labeled with Cy5 and intravenously injected (i.v.) after i.t. PBS or i.t. E. coli. The IVIS image revealed that OT-I CD8 T cells infiltrated into tumor tissues at a higher quantity in the E. coli group (Fig. 2c). Tumors were harvested and quantified for Cy5 intensities after 24 h post-treatment. The Cy5 intensities in the E. coli group were nearly 1.57-fold of that in the PBS group (Fig. 2d). We leveraged flow cytometry to quantify the infiltration increment precisely. As a result, the proportion of the infiltration OT-I T cell fraction of tumor tissues in the E. coli group was ~35.3-fold of that in the PBS group (Fig. 2e). We initially tested whether the improved tumor accumulation of tumor-specific T cells could bring therapeutic advantages. We adopted OT-I T cells and anti-hCD19 murine CAR-T cells for B16F10-OVA and Panc02-hCD19-luci tumor treatment, respectively (Supplementary Fig. 6).^33,34 As a result, this combination achieved 87.5% complete tumor remission in melanoma and significantly inhibited pancreatic tumor growth (Fig. 2f, g). Importantly, the mice’s body weight was kept in the normal range in both experiments (Supplementary Fig. 7). In addition, we also monitored the long-term survival of this combination strategy for murine melanoma treatment. E. coli + OT-I group achieved a 6/7 survival rate up to 40 days (Supplementary Fig. 8). Routine blood tests, serological analysis, and H&E images showed no significant variations compared to the healthy mice at the same age (Supplementary Figs. 9, 10).

E. coli MG1655 reprograms TAM to secrete CCL5 for recruiting the adoptive transferred T cells

We further delved into elucidating the specific mechanisms underpinning the enhancement of T cell tumor infiltration efficacy mediated by intratumor bacterial therapy. The initial response of the endogenous innate immune system to exogenous bacterial invasion holds paramount importance in this process.³⁵ During this intricate interplay, several crucial chemokines, including CCL3, CCL4, and notably CCL5, have been identified as key players in orchestrating T cell recruitment towards the tumor microenvironment.³⁶ Drawing upon the comprehensive data gathered from Luminex assay analysis (Fig. 1b and Supplementary Fig. 1), we decided to focus our investigations on the CCL5 signaling axis. This choice was predicated on the significance of CCL5 in directing T cell migration and its potential to serve as a pivotal mediator in augmenting the efficacy of intratumor bacterial therapy.³⁷

We initially conducted co-culture experiments between bacteria and bone marrow-derived macrophages (BMDM), discovering that E. coli MG1655 significantly upregulated the expression of M1-phenotype markers in BMDM (Fig. 3a). This finding suggests a shift towards an inflammatory macrophage phenotype induced by E. coli. Furthermore, in the context of tumor-associated macrophages, we observed an elevated level of CD86 expression in the presence of E. coli within the OT-I group, compared to the OT-I group alone (Fig. 3b). Moreover, the addition of E. coli notably boosted BMDM to secrete CCL5 (Fig. 3c).³⁸ To understand the potential role of CCL5 in this setting, we analyzed the expression of the receptor of CCL5, CCR5, on OT-I CD8 T cells using flow cytometry (Supplementary Fig. 11). For functionally assessing the impact of E. coli-induced CCL5 on T cell infiltration, we employed a transwell migration assay.³⁹ Our results demonstrated that the supernatant of the coculture (E. coli with BMDM) led to the highest level of OT-I CD8 T cell transmigration (Fig. 3d). Furthermore, the ability to block T cell infiltration through the use of the CCL5 neutralization antibody underscores the critical role of CCL5 in directing the migration of OT-I CD8 T cells towards the tumor site.

Furthermore, we explored the impact of the tumor-associated macrophage (TAM) depletion agent, clodronate liposome, and CCL5 neutralization antibody, on the accumulation of OT-I CD8 T cells within the tumor tissues (Fig. 3e). Our flow cytometry analysis revealed that both treatments significantly reduced the proportion of intratumor OT-I T cells. However, it is noteworthy that the extent of downregulation was less pronounced in the CCL5 antibody group compared to the clodronate liposome group. This observation could potentially be attributed to the presence of additional CCL5-independent chemotaxis players that contribute to T cell infiltration within the tumor.⁴⁰ To assess the therapeutic implications of the reduced OT-I T cell infiltration induced by clodronate liposome or CCL5 neutralization antibody, we conducted an analysis of tumor growth inhibition (Fig. 3f). Notably, the tumor inhibitory effect was substantially diminished in the clodronate liposome group compared to the E. coli group, indicating the importance of TAMs in facilitating T cell-mediated tumor suppression. The mean tumor volume at the end of the monitoring time point was the lowest in the E. coli + OT-I T cells group, mirroring the critical role of both TAMs and CCL5 in mediating T cell infiltration for potentiating the antitumor immune response.

E. coli adjuvants transferred T cells for distal tumor control through in situ tumor vaccination

Given that E. coli present a diverse array of pathogen-associated molecular patterns, they possess the capability to prime the innate immune system, which in turn can stimulate adaptive immune responses directed against pathogens or, in this context, tumor cells.⁴¹ To investigate the impact of E. coli on intratumor immune cell phenotypes, we conducted flow cytometry analysis (Fig. 4a). Our findings revealed that the combination of E. coli and OT-I T cells elicited the highest levels of intratumor immune cell infiltration (Fig. 4b). Notably, both the E. coli group and the E. coli + OT-I T cell group significantly reduced the proportion of M2-like macrophages (Fig. 4c) and induced the maturation of intratumor dendritic cells, as evidenced by increased expression of CD80 and CD86 (Fig. 4d). Furthermore, to gain insights into the underlying mechanisms mediating the crosstalk between innate and adaptive immunity, we utilized a Luminex assay to quantify intratumor chemokine and cytokine levels. Our results indicated that the addition of E. coli successfully reinvigorated the tumor microenvironment compared to OT-I T cells alone, as demonstrated by the upregulation of various chemokines and cytokines essential for immune cell recruitment, activation, and effector function (Fig. 4e).^42,43 These findings underscore the pivotal role of E. coli in orchestrating the crosstalk between innate and adaptive immunity, thereby potentiating antitumor immune responses.

Building upon our previous findings, we extended our investigation to a double-flank melanoma mouse model. This model could partially offer a valuable tool to assess the therapeutic potential of locally administered treatments in influencing tumor growth at distant sites or metastasis, providing insights into their systemic effects and potential for controlling disease progression beyond the primary tumor location. Specifically, when the larger tumor reached approximately 50 mm³, we administered E. coli and OT-I T cells (Fig. 4f). Our results demonstrated that the E. coli + OT-I group exhibited remarkable therapeutic efficacy, achieving tumor eradication in 4 out of 7 in primary tumors and 3 out of 7 in distal tumors (Fig. 4g, h). Notably, we observed that E. coli alone was also capable of inhibiting the growth of distal tumors, prompting us to investigate deeper into the mechanisms underlying this distal tumor-restraining effect. To explore these mechanisms, we focused on the potential of E. coli to enhance the tumor antigen presentation capabilities of dendritic cells (DCs), which are crucial for priming tumor-specific T cells that can mediate distal tumor control. Flow cytometry analysis revealed that the E. coli + OT-I group had the highest proportion of H-2Kb SIINFEKL-presenting DCs (Supplementary Fig. 12a).⁴⁴ This, coupled with the pro-maturation effects of E. coli as an adjuvant (Fig. 4d), suggests that E. coli can elicit robust endogenous antitumor immune responses. Furthermore, we observed that the activation state of circulating CD8 T cells was significantly enhanced in both the E. coli and E. coli + OT-I groups (Supplementary Fig. 12b). This finding underscores the ability of E. coli to stimulate systemic immune responses that extend beyond the primary tumor site, contributing to the observed distal tumor-restraining effect.

Combination treatment eradicates advanced-stage melanoma and hepatocellular carcinoma

Given the promising therapeutic efficacy and notable tumor regression observed in preclinical models of small tumors, we further explored the therapeutic potential of this combined treatment approach in advanced-stage tumors. For this purpose, we generated TA99 murine CAR-T cells that could specifically recognize melanoma-associated tyrosinase-related protein 1 (TRP-1) and rigorously characterized their phenotypic profile (CD4/CD8 ratio and CCR5 expression) as well as their ability to specifically eliminate B16-luci tumor cells in vitro (Supplementary Fig. 13).^45,46 When applied to murine melanoma models where tumor volumes had reached 300 mm³, the combination of E. coli with TA99 CAR-T cells resulted in a remarkable 50% complete tumor remission rate (Fig. 5a–c). Furthermore, the therapeutic regimen demonstrated satisfactory tolerability, with no significant adverse effects on mouse body weight or temperature throughout the treatment period (Fig. 5d–f). For the treatment of hepatocellular carcinoma, we applied a combination of E. coli and OT-I CD8 T cells once the tumor volume reached 400 mm³ (Fig. 5g).⁴⁷ Remarkably, the E. coli + OT-I T cell group achieved a tumor-free rate of 100% (Fig. 5h, i). Notably, all mice in this group remained alive for 15 days post-treatment (Fig. 5j). The body weights of mice in the E. coli + OT-I T cell group kept within the normal range throughout the treatment period, indicating the satisfactory tolerability of this therapeutic approach (Fig. 5k).

Spatial cooperation between E. coli and transferred T cells eliminates solid tumor

Given the exceptional anti-tumor efficacy demonstrated in advanced-stage solid tumors, we started an investigation to elucidate the underlying mechanisms of this therapeutic strategy. To this end, we initiated the treatment modalities once the tumors had grown to 400 mm³ (Fig. 6a). As a result, the tumor volume in the combinatory treatment group (E. coli + OT-I) decreased significantly, in stark contrast to the continued growth observed in all other treatment groups (Fig. 6b). Importantly, the body weights of the mice remained stable and within the normal range throughout the treatment period, indicating good tolerability (Fig. 6c). Notably, five out of eight mice in the E. coli + OT-I group achieved a tumor-free state, further underscoring the potency of this combined therapeutic approach (Fig. 6d, e). To gain a deeper understanding of the mechanisms underlying the remarkable therapeutic efficacy observed, we performed histological and immunohistochemical analyses of the tumors using hematoxylin and eosin (H&E) staining, Ki-67 proliferation marker, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Specifically, we observed a necrotic area in the core of tumors treated with E. coli alone, the tumor periphery in the OT-I group, and a nearly complete region in the E. coli + OT-I combinatory group (Fig. 6f).⁴⁸

Further analysis with Ki-67 and TUNEL staining revealed a reversed location signature, with decreased proliferation (Ki-67) and increased apoptosis (TUNEL) in the E. coli + OT-I group, mirroring the trends observed in the H&E images (Fig. 6g, h). These findings are consistent with previous evidence suggesting that intratumor bacterial therapy can delay tumor growth but may be insufficient to control the rapid outgrowth of the tumor periphery.⁴⁹ However, our results demonstrate that the addition of OT-I CD8 T cells to the E. coli therapy significantly enhances its efficacy, resulting in a more comprehensive and effective tumor regression. Based on our findings, we hypothesize that intratumor administration of E. coli is capable of effectively destroying the hypoxic inner core of solid tumors but may be limited in its ability to restrain tumor growth at the periphery. In contrast, adoptive T cell therapy exerts a more localized, albeit less potent, therapeutic effect primarily focused on the tumor periphery.⁵⁰ The combination of these two therapies, however, not only directly targets and destroys the inner tumor core but also significantly enhances the infiltration and tumor killing of T cells within the tumor periphery. To strengthen our hypothesis, we located intratumor T cells and E. coli (Fig. 6i). E. coli was discovered to colonize primarily within the hypoxic inner core of the tumor. In the OT-I group, T cells were localized primarily at the tumor periphery.⁵¹ However, in the combination therapy group, we observed a significantly higher quantity of T cells infiltrating the tumor tissues, ultimately leading to complete control of advanced-stage solid tumor growth throughout the entire tumor area. These findings further support our hypothesis that the combination of intratumor E. coli and adoptive T cell therapy offers a synergistic and potent approach for the treatment of advanced-stage solid tumors.

Intratumor bacterial therapy promises therapeutic biosafety

One significant concern regarding the use of bacteria in therapeutic applications is the potential for toxicity stemming from bacterial infections.⁵² To ensure the safety and efficacy of bacterial therapy, it is crucial to achieve successful colonization of bacteria within tumor tissues, which necessitates careful optimization and testing of the bacterial dosage. In our study, we employed LuxCDABE-transduced E. coli MG1655 to monitor the colonization of intratumor E. coli. Our results indicated that the bioluminescence of E. coli declined rapidly within the initial 12 h and then remained stable for at least five days (Supplementary Fig. 14). On the endpoint day, we confirmed that the majority of E. coli were confined to the tumor tissues, with minimal impact on other major organs (Supplementary Fig. 15). To assess the potential for bacterial infection to induce a cytokine storm, we monitored both intratumor and serum cytokine levels throughout the therapeutic procedure. Notably, all inflammatory cytokine markers, including IFN-γ, TNF-α, IL-1β, IL-6, and IL-10, exhibited a transient increase in serum samples immediately after the initial bacterial injection. However, these levels rapidly returned to normal ranges, similar to pre-injection levels (Supplementary Fig. 16). This suggests that our bacterial therapy was well-tolerated and did not induce severe systemic inflammation, highlighting the potential for safe and effective bacterial-based therapies in the treatment of tumors. We further performed routine blood and serological tests two days post-treatment. The white blood cell, lymphocyte, neutrophil, and red blood cell were all in the normal range (Supplementary Fig. 17). The neutrophil exhibited higher in the E. coli and the E. coli + OT-I groups.⁵³ No significant change was observed in creatinine (CRE-J), total bilirubin (TBIL), and urea (Supplementary Fig. 18). All the treatment groups except the PBS group effectively downregulated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentration.

SEO Powered Content & PR Distribution. Get Amplified Today.
PlatoData.Network Vertical Generative Ai. Empower Yourself. Access Here.
PlatoAiStream. Web3 Intelligence. Knowledge Amplified. Access Here.
PlatoESG. Carbon, CleanTech, Energy, Environment, Solar, Waste Management. Access Here.
PlatoHealth. Biotech and Clinical Trials Intelligence. Access Here.
Source: https://www.nature.com/articles/s41392-024-02028-3

Remolding the tumor microenvironment by bacteria augments adoptive T cell therapy in advanced-stage solid tumors

Intratumor E. coli modulates tumor physical and physiochemical microenvironment

Intratumor E. coli treatment augments OT-I T cell infiltration into solid tumors

E. coli MG1655 reprograms TAM to secrete CCL5 for recruiting the adoptive transferred T cells

E. coli adjuvants transferred T cells for distal tumor control through in situ tumor vaccination

Combination treatment eradicates advanced-stage melanoma and hepatocellular carcinoma

Spatial cooperation between E. coli and transferred T cells eliminates solid tumor

Intratumor bacterial therapy promises therapeutic biosafety

Latest Intelligence

How clinical trial diversity is the path to health equity

Biotech startups are built on venture capital. Track funding rounds here.

Novartis wagers more than $1B on gene therapies for the nervous system

Versant enters obesity with new startup

Huntington’s disease: a therapeutic field on a bumpy ride

Eight areas where gene therapy shines

Boshoff to succeed Dolsten as Pfizer CSO

Helping diabetic wounds heal

Science Spotlight: A new obesity target may avoid nausea, muscle loss

Raining on biotech’s parade — the mood from London: a Perspective