Programmable bacteria act as live biotherapeutics to remodel tumor stroma and potentiate immunotherapy

doi:10.21203/rs.3.rs-5070038/v1

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Programmable bacteria act as live biotherapeutics to remodel tumor stroma and potentiate immunotherapy

https://doi.org/10.21203/rs.3.rs-5070038/v1

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Immunotherapy has transformed cancer treatment but its efficacy is limited in many solid tumors in large part due to the immunosuppressive tumor microenvironment (TME). TME might be composed of a dense/stiff extracellular matrix (ECM) that impedes infiltration of immune cells. To address this abnormality, Neobe Therapeutics developed an engineered live biotherapeutic in the form of programmable bacteria that offer advantages for delivering enzymes to remodel the ECM. Using engineered Escherichia coli to express and release hyaluronidase under a hypoxia-inducible promoter, the bacteria target hyaluronan within the TME. Our study demonstrates that hyaluronan degradation reduces tumor stiffness and restores vascular functionality in murine models of breast cancer. This potentiates immune checkpoint inhibitor efficacy by facilitating immune cell infiltration and immunostimulation. Additionally, our findings suggest that the potency of antitumor responses depends on baseline stiffness levels and ECM composition, highlighting the potential of programmable bacteria to improve immunotherapy outcomes in drug resistant, solid tumors.

Biological sciences/Cancer/Cancer microenvironment

Biological sciences/Biotechnology/Tissue engineering

immune checkpoint inhibition

live biotherapeutics

tumor perfusion

stroma normalization

shear wave elastography

Cancer immunotherapy harnesses the body's immune system to combat cancer. Since the approval of ipilimumab as the first FDA-approved immune checkpoint inhibitor (ICI) in 2010 ¹, cancer immunotherapy has emerged as a major therapeutic avenue, revolutionizing the treatment of multiple hematological and solid malignancies. However, its therapeutic efficacy varies considerably, with a substantial fraction of patients experiencing low response rates and a significant incidence of immune-related adverse events (irAEs) ². Response rates to ICIs range from 10-60% with initial treatment, and many initially responsive patients develop secondary resistance over time ^3,4. The limited efficacy of immunotherapy is in large part attributed to the pre-existing immunosuppressive tumor microenvironment (TME). Indeed, in solid tumors, such as subtypes of breast, colon, pancreatic and prostate cancers, the tumor becomes stiff as it grows within the host's normal tissue. Tumor stiffening arises from an abnormal increase in structural components of the tumor, particularly the extracellular matrix (ECM). In turn, the accumulation of a dense/stiff ECM exerts mechanical forces, leading to vascular compression, hypo-perfusion and compromised tissue oxygenation. Consequently, the systemic administration of drugs, such as ICIs, as well as the recruitment, infiltration and activation of cytotoxic immune cells are hindered ^5-9.

To overcome these barriers, recent studies, have shifted focus towards repurposing clinically approved drugs, known as mechanotherapeutics, targeting one or more factors contributing to tumor tissue stiffening and TME mechanical abnormalities ^10-16. However, direct targeting of the ECM via drug repurposing presents limitations as adding a new drug to the treatment regimen of cancer patients is often not feasible due to dose-limitation restrictions and the risk of adverse events stemming from prolonged administration. This is exemplified by the discontinuation of PEGPH20, a nanoparticle formulation of recombinant human hyaluronidase. Although PEGPH20 demonstrated promising results in preclinical studies by improving ICI and chemotherapy outcomes ^17,18, it failed in phase 3 trials due to off-target toxicity issues, necessitating significant dose reductions that ultimately proved to be clinically ineffective ¹⁹. The angiotensin receptor blocker and common anti-hypertensive losartan was successfully repurposed to modulate collagen and hyaluronan levels in human pancreatic tumors and increase resection rates following chemo-radiation treatment²⁰. However, many patients were excluded from the trial due to the potent anti-hypertensive effects of losartan and losartan recently failed to improve the efficacy of ICI in a follow-up multi-center phase II trial²¹. To date, no other approaches involving the direct breakdown of fibrotic barriers to immune cell infiltration have progressed to phase 3 trials, underlining the urgent need for more targeted and safer delivery systems.

As an alternative to this approach, Neobe Therapeutics has developed a synthetic biology platform to generate live biotherapeutics in the form of engineered bacteria that are exploited for the delivery of anticancer agents as well as antiangiogenic and stroma-targeting compounds ^22,23. The unique properties of the TME, such as low oxygen levels, an immunosuppressive environment, and the release of nutrients from necrotic cells favor the accumulation and colonization of facultative and obligate anaerobes. Even a small fraction of the bacterial dose reaching the tumor is sufficient to colonize it and allow for the sustained release of therapeutic payload over an extended period without harming healthy tissues.

In this study, a central part of Neobe’s platform leveraged the acidic and hypoxic conditions commonly found within tumors to develop a live biotherapeutic product in the form of programmable bacteria. This product serves as a delivery platform for enzymatic degradation of hyaluronan (HA) within the TME, aiming to reduce stiffness and restore perfusion and oxygenation in solid tumors. Specifically, Neobe engineered safe strains of tumor colonizing bacteria to express the enzyme hyaluronidase under a hypoxia-inducible promoter. Our findings demonstrate that HA degradation reduces intratumoral stiffness levels and restores vascular functionality in murine models of triple-negative breast cancer. This engineering of the TME, significantly enhances the efficacy of ICI in tumors refractory to treatment by overcoming the physical barriers to immune cell infiltration and promoting immunostimulation. Moreover, by exploring the ICI combination using a less desmoplastic tumor model, we highlight the dependence of the potency of antitumor responses on baseline stiffness levels and ECM composition.

Engineered E. coli expressing bacterial hyaluronidase effectively degrades hyaluronan acid in human tissues

E. coli were genetically engineered to express the bacterial hyaluronidase by cloning the PelB signal sequence at the N-terminus of hyaluronidase (HAse) coding sequence, under the control of a rhamnose-inducible promoter, generating the E. coli-HAse bacterial strain. We determined the growth kinetics of E. coli-HAse expressing strains over 24 hours in non-induced and induced (0.2% rhamnose) conditions (Supplementary Fig. 1a). The HAse expression and activity were assessed by incubating E. coli-HAse in LB media, supplemented with 0.4 mg/ml purified hyaluronan in the presence and absence of rhamnose. LB media supplemented with 0.4 mg/ml purified hyaluronan was used as a negative control. The OD600 in each condition was measured over 6 h. Our findings show that under induced conditions, E. coli-HAse is capable of degrading purified hyaluronic acid, as demonstrated by cetyltrimethylammonium bromide turbidimetric method (CTM ) (Supplementary Fig. 1b). As the expressed Hyaluronidase contained a Flag tag, we were able to detect it by western blot in both bacterial pellets and conditioned media, confirming effective enzyme release (Supplementary Fig. 1c). Consistent with this, the supernatant collected from induced E. coli-HAse effectively degrades hyaluronic acid, whereas no HA degradation was observed in the supernatant collected from uninduced bacterial culture when compared to a control engineered strain expressing GFP under the same promoter (Supplementary Fig. 1d).

Next, we sought to examine the HAse ability to break down human hyaluronan in its native form. For this, we tested bacteria activity in tumor samples obtained from breast cancer patients, both as 2D tissue sections and 3D tissue explants. Tumor tissue explants from patients with triple-negative breast cancer were decellularized as previously described, in a process that is shown to maintain the structure, heterogeneity and composition of the ECM ²⁴. E. coli-HAse was incubated with decellularized tissue explant material in the presence or absence of the hyaluronidase expression inducer molecule, rhamnose. After 4 h, the explant material was stained with biotylinated hyaluronic acid binding protein and imaged by light microscopy, showing that explants incubated with E. coli-HAse lose almost all hyaluronic acid content. E. coli-HA was further added to 2D tumor tissue sections at increasing CFU doses, showing dose-dependent degradation with maximum degradation occurring at the 10⁷ CFU dose (Supplementary Fig. 1e-g).

E. Coli-Hase facilitates hyaluronic acid degradation through tumor-specific colonization

To validate the tumor-specific colonization of the programmable bacteria, we engineered a bioluminescent version of Neobe’s E. Coli bacterial chassis strain, constitutively expressing luciferase using the luxCDABE operon ²⁵. This E. Coli-lux engineered strain enabled us to monitor the biodistribution of Neobe’s E. Coli chassis in the murine 4T1 triple-negative breast cancer model. Bacteria were administered at various doses (10⁶, 10⁷ and 10⁸ CFUs) via a single intravenous injection, and mice were imaged at 24, 48 and 72 h post-administration using bioluminescence in vivo imaging. Mice administered the bioluminescent E. coli strain at doses of 10⁶ CFU and 10⁷ CFU maintained mean body weights comparable to those of mice administered the vehicle alone, while the higher dose of 10⁸CFU per mouse caused greater weight loss (Supplementary Fig. 2a, b). Importantly, all doses exhibited tumor-specific colonization with the highest dose being evident as early as 24 h. Given the weight loss in the group administered 10⁸ CFU of the candidate strain, the level of colonization in the tumor and secondary organs was quantified in mice administered a dose of 10⁷ CFU at 72 h post-administration. Luminescence was localized in the tumor and CFU counting showed a high concentration of bacteria in the tumor (10⁸ CFU/g tissue) with residual levels of bacteria in secondary organs (around or below 10⁴ CFU/g tissue, Supplementary Fig. 2c). These findings demonstrate that Neobe’s E. coli chassis is well-tolerated and localizes to and accumulates in tumor tissue.

Having confirmed the tumor colonizing ability of the chassis strain at well-tolerated doses, we proceeded to test the efficacy of the hyaluronidase-expressing strain in remodeling the tumor microenvironment. To ensure that payload expression is contained within the tumor, two different variants of the E. Coli-HAse different strains were generated expressing hyaluronidase under hypoxia-inducible promoters (C35R7 and C35R9) and tested together with an additional variant expressing hyaluronidase under a constitutive promoter (phelp). After confirming the tumor-specific colonization, we sought to investigate the potential of the three variants in remodeling the TME through HA degradation in orthotopic 4T1 tumors that are rich in HA and other ECM components ^26,27. Tumor-bearing mice were intravenously injected with either phelp, C35R7 or C35R9 bacterial constructs at a dose of 10⁶ and 10⁷ CFUs, to identify the most effective candidate and dose for remodeling the TME. Changes in HA content were evaluated by immunofluorescence staining using hyaluronan binding protein (HABP) following tumor removal 7 days after bacterial administration (Fig. 1a). Interestingly, our data demonstrate that only the low dose of C35R7 can significantly reduce HA levels in 4T1 tumors, as indicated by the 2-fold reduction in HABP signal (Fig. 1b). As expected, none of the bacterial constructs had an antitumor effect, nor did they cause a reduction in body weight exceeding the 20% cutoff compared to the control group. The minimal weight loss observed the day after bacterial treatment is attributed to the shock to which mice were subjected to (Supplementary Fig. 3a, b). Nevertheless, we observed that administration of the high dose of phelp and C35R7 live biotherapy could cause spleen enlargement, potentially as an acute reaction to the initial bacterial dose (Supplementary Fig. 3c).

HA degradation leads to reduction of tumor stiffness and restoration of perfusion

HA abundance in desmoplastic tumors determines in large part the tumor’s mechanical microenvironment by inducing tissue stiffening, which promotes vessels collapsing and hypo-perfusion ^26-28. To assess the role of the engineered bacteria in remodeling the TME, we employed the 4T1 tumor model and monitored in vivo the changes in the TME’s mechanical properties using ultrasound shear wave elastography (SWE) imaging. SWE measures tissue stiffness in real-time by generating a color map indicating different magnitudes of elastic modulus in kPa. SWE was performed before bacterial administration to establish baseline values, after 3 days of administration and at the end of the study (Fig. 2a, b). Interestingly, our findings demonstrate that the higher dose of phelp-HAse (10⁷ CFUs) and both doses of C35R7-Hase (10⁶and 10⁷ CFUs) can significantly decrease tumor stiffness, compared to the control group, as early as 3 days post-administration with the effect sustained even after 7 days, regardless of the dose employed (Fig. 2c). On the contrary, C35R9-HAse did not have any effect on tumor stiffness at either of the two doses administered in agreement with its inability to reduce HA levels, suggesting that either this promoter may be less effective at inducing Hase expression, or might exhibit a slow release of HAse, requiring more time to see an effect.

Given that alleviation of tumor stiffness is associated with improved perfusion across solid tumors ²⁹, we investigated whether the bacteria could also improve vascular perfusion of 4T1 tumors. Tumor perfusion was assessed using contrast-enhanced ultrasound (CEUS) following a bolus injection of contrast agents (microbubbles). CEUS was employed twice: on the day of bacterial administration and at the study endpoint (Fig. 2a). Analysis of the ultrasound images of contrast agents at the time of peak intensity indicates that only the high concentration of phelp-HAse (10⁷ CFUs) can enhance perfusion compared to the untreated group, while C35R7-Hase is beneficial at any of the doses (10⁶and 10⁷ CFUs) employed (Fig. 2d,e). Moreover, we confirmed the existence of a linear correlation between tumor stiffness and perfusion values as measured at the end of the study consistent with previous studies (Fig. 2f)^10,29. Considering its limited toxicity and its ability to effectively reduce HA levels and remodel the TME, we concluded that C35R7 at a dose of 10⁶ CFUs is the ideal live biotherapeutic to continue our research with.

C35R7 enhances immunotherapy efficacy in triple-negative breast cancer murine models

The accumulation of HA in the TME serves as a physical barrier, compromising the delivery of ICIs and restricting access of immune cells to the tumor interior, highlighting the significance of ECM targeting as a potential approach to enhance the efficacy of immunotherapy. To address this, we conducted combination studies involving bacterial-mediated enzymatic stromal remodeling using the C35R7-Hase candidate and an antibody cocktail consisting of anti-PD-1 (10 mg/kg) and anti-CTLA-4 (5 mg/kg) in two orthotopic syngeneic models of breast cancer, namely 4T1 and E0771, which are known to be refractory to ICI^26,27,30-32. 4T1 and E0771 tumor-bearing mice received the bacterial treatment only once when tumors reached an average size of 150 mm³. Given that bacteria can reduce stiffness as early as 3 days post-administration, the first cycle of the immunotherapy cocktail was administered 3 and 4 days post-bacterial treatment in 4T1 and E0771 studies, respectively. In total, mice received three cycles of the immunotherapy cocktail (via intraperitoneal injection) with a three-day interval between each cycle. Interestingly, for the 4T1 study, only mice treated with the combination therapy experienced tumor regression, while ICI monotherapy had no effect on tumor growth (Fig. 3a,c and Supplementary Fig. S4a). In the E0771 study, ICI alone had a modest but significant antitumor effect consisting of an initial plateauing of the tumor growth rate, which eventually resumed (p=0.036), while the combination of C35R7 with ICI exhibited a more potent antitumor response (p=0.0008), with 5 out of 10 mice in that group experiencing complete tumor regression (Fig. 3b, d and Supplementary Fig. S4b,c). To validate that the enhanced antitumor responses were attributed to the remodeling of the TME, we performed SWE and CEUS, before and 3 or 4 days after bacterial treatment, as described earlier. Consistent with our previous results, we confirmed that the groups receiving the bacteria had reduced intratumoral stiffness levels (Fig. 3e, f) and increased vascular perfusion compared to the tumors of groups treated with either ICI monotherapy or left untreated (Fig. 3g).

Combination treatment prolongs survival in triple-negative breast cancer models

The effectiveness of the engineered bacteria to boost immunotherapy efficacy was further supported by survival studies (Fig. 4a,b). To mimic the clinical scenario, tumors were surgically removed 3 days after the last ICI cycle and mice survival was closely monitored. For the 4T1 tumor model, our data demonstrate that 1 out of 9 mice treated with C35R7 + ICI achieved complete remission (Fig. 4a). For the E0771 study, mice of the untreated group were the first to die followed by the mice administered with only the bacteria 10 days apart, although the survival advantage noted was not statistically significant. One mouse out of 9 treated with ICI monotherapy experienced complete survival, while all mice of the combination treatment were cured (Fig. 4b, c). Survivors of both ICI monotherapy and the combination group were rechallenged with a second inoculation of E0771 cells and their growth curves were compared against 8 healthy control mice of the same age. Remarkably, tumors developed in all control mice (Fig. 4d) but none in the immunotherapy and combination-treated mice that survived the initial experiment (Fig. 4e, f), indicating the acquisition of durable long-term antitumor response and immunological memory to E0771 cancer cell antigens.

Programmable bacteria combined with ICI immunostimulates the TME

Given the pivotal role of the tumor immune microenvironment in determining sensitivity to ICI treatment, we aimed to examine the immune milieu of tumors following various treatments. Flow cytometry analysis revealed a reduction in the myeloid-derived suppressor cell (MDSC, CD45⁺GR1⁺CD11b⁺) population in both 4T1 and E0771 tumors treated with ICI alone or the combination therapy, whereas the levels of total lymphocyte remained unchanged across the different treatment groups (Fig. 5a, b, h, i). Consistent with this finding, the increase in the percentage of tumor-associated macrophages (TAMs, CD45⁺GR1^-CD11b⁺F4/80⁺) and the elevation in the ratio of M1-like TAMs to M2-like TAMs, in the TME of E0771 tumors treated with ICI combination further suggests a shift from an immunosuppressive to an immunosupportive microenvironment (Fig. 5j, k). Furthermore, E0771-bearing mice treated with immunotherapy exhibited enhanced infiltration of helper and cytotoxic T cells (Fig. 5l, m), while the ratio of CD8⁺ T cells to immunosuppressive regulatory T cells (Tregs, TCRB⁺CD4SP⁺CD25^hiCD127^loFoxp3⁺) remained unaffected (Fig. 5n). The lack of statistical significance in the ICI combination group of the E0771 study is likely attributed to the small sample size. In the 4T1 study, we did not observe any changes in the abundance of TAMs or T cells, despite an increase in the CD8 T cell/Tregs ratio following ICI monotherapy. This lack of change is likely attributed to the fact that immunophenotyping of tumors was analyzed at the study endpoint, and might have missed detecting immune cell infiltration and activation during the earlier phase of the immune response. (Fig. 5c-g).

Antitumor potency of ICI combination therapy is dependent on baseline stiffness levels

Considering the positive impact of C35R7 live biotherapeutic in potentiating immunotherapy effectiveness in breast cancer, we wondered whether a less fibrotic tumor could respond similarly to the combined treatment. To address this, we employed the CT26 orthotopic colorectal cancer model by engrafting a small CT26 tumor fragment onto the caecum of mice, following established protocols ³³. Mice were allowed to recover from surgery and then treated with a single i.v. injection of C35R7 at a concentration of 10⁶ CFUs, as described earlier. Importantly, we observed that systemic administration of bacteria could effectively degrade HA levels in the CT26 tumor stroma without having any antitumor effects (Supplementary Fig. 6a-c), although the reduction noted was to a lesser extent compared to the 4T1 tumor model (Fig. 1). No significant adverse events including animal weight loss or splenomegaly were observed due to bacterial treatment (Supplementary Fig. 6d, e).

Having confirmed the enzymatic degradation of ECM in CT26 colon tumors, we next proceeded to ICI combination studies. Specifically, we employed a CT26 orthotopic tumor model stably expressing the luciferase reporter (CT26-Luc), allowing for real-time monitoring of tumor growth via bioluminescence imaging (BLI). Once the bioluminescence signal was detectable across all study mice (i.e., day 5 post-implantation), the bacterial treatment was administered. Then, 3 days after the C35R7 injection, mice received the first cycle of ICI cocktail, followed by two additional cycles. Photon flux (photons/sec) was used to quantify the bioluminescence signal, serving as an indicator of tumor progression. Notably, we observed a similar reduction in mean tumor burden in mice treated with the ICI alone or C35R7-Hase plus ICI combination (Fig. 6a-c). Individual BLI curves indicate that mice start responding to immunotherapy after the second cycle of treatment (day 11), with more mice in the combination group exhibiting a complete tumor regression compared to the monotherapy group (Fig. 6d-g), although not statistically significant. To assess whether the responsiveness of CT26 tumors to ICI is also reflected in the composition of the immune tumor microenvironment, we examined the prevalence of different immune cell populations following bacterial or ICI monotherapy, bacterial+ICI combination therapy, and in untreated mice. The intratumoral lymphocyte population remained unaffected across different treatment groups, consistent with our previous study in the triple-negative breast cancer models (Fig. 6h). Surprisingly, unlike the 4T1 and E0771 tumors, the levels of myeloid cells did not decrease after immunotherapy treatment, suggesting a tumor type-specific mechanism of action for ICI (Fig. 6i, j). T cell infiltration increased in groups receiving the ICI cocktail, but no change in CD8⁺T cell to Tregs ratio was observed (Fig. 6k, l). Finally, we assessed the levels of antigen-presenting cells and confirmed their upregulation after ICI treatment (Fig. 6m). To support our hypothesis that ECM enzymatic degradation combined with ICI is more likely to benefit highly desmoplastic tumors refractory to ICI monotherapy, we assessed the tissue elasticity of CT26-luc tumors and compared them with the reported elastic moduli of 4T1 and E0771 tumors. This comparison was conducted through ex-vivo stress-strain experiments ³⁴, as the in vivo measurement of CT26 tissue stiffness using SWE was not feasible with our ultrasound system. Interestingly, the ex-vivo elastic modulus measurements indicated that CT26 colon tumors are significantly softer compared to the triple-negative breast cancer models employed in the current study (Supplementary Fig. 7a). Immunofluorescence staining for the major ECM components HA and collagen confirms this finding (Supplementary Fig. 7b).

The vast majority of currently under-development solutions for remodeling the stroma of solid tumors focuses on targeting cancer-associated fibroblasts (CAFs) which are the main contributors to ECM production. However, this strategy poses several challenges due to the high heterogeneity of CAF population within the TME and across different types of solid tumors ³⁵. Moreover, the degradation of ECM components often extends beyond the tumor itself, affecting healthy tissues that share these components due to the lack of specificity. To address these limitations, in this study, we exploited genetically engineered bacteria as carriers for delivering therapeutic payloads. Extensive evidence shows that bacteria are both inherently present and selectively grow within tumors, making them a natural platform for creating customizable therapeutic solutions. This capability is attributed to reduced immune surveillance and their ability to thrive in hypoxic environments ^36,37. The primary mechanism by which engineered bacteria can contribute to antitumor responses is via the release of cytotoxic compounds resulting in local cancer cell death and impeding further tumor growth. Secondly, they act as potent immune stimulators, triggering an immune response against the tumor.

To date, several types and strains of bacteria such as Listeria, Salmonella and Clostridium species, have been found to be well-suited for delivering therapeutic payloads ^22,23,38. Nevertheless, the utilization of such pathogenic bacteria prerequisites attenuation to weaken their virulence, rendering them no longer threatening to the host organism, while retaining their tumor-targeting capacity. On the contrary, commensal microorganisms represent a safer alternative to attenuated bacteria. Due to their long-established presence in the human microbiome are better tolerated by the immune system, reducing the risk of adverse reactions. Thus, their use is less likely to raise safety concerns associated with reversion to virulence. Specifically, E. coli is a prime candidate for bioengineering due to its historical use as an oral probiotic and its suitability for diverse applications ³⁹.

Using the bacterial chassis E. Coli K12 MG1655,Neobe Therapeutics employed their synthetic biology platform to engineer three live biotherapeutic prototypes expressing hyaluronidase under different promoter systems, phelp, C35R7 and C35R9, the latter two of which being tumour-inducible promoters which enable tumor-specific expression and secretion directly into the extracellular space. Biodistribution studies of luciferase-expressing E. coli and quantification of bacterial load in various organs confirmed the preference of this chassis for colonizing 4T1 tumors. The capability of the engineered bacterial prototypes to degrade the ECM was validated in vivo using preclinical models of breast cancer. Of the three bacterial constructs tested, we show that only the low dose (10⁶ CFUs) of C35R7 bacterial treatment could decrease the HA levels by 50% in the 4T1 tumors with a statistically significant difference, compared to the untreated mice. On the contrary, higher doses (10⁷ CFUs) of bacteria were ineffective suggesting that other secondary responses may be triggered leading to ECM compensation because of the excessive HAse production. Our findings demonstrate that reduction in HA levels suffices to decrease the stiffness of the tumor tissue facilitating vessel decompression and restoring perfusion. Consistent with the function of mechanotherapeutics targeting the ECM^{10,26,27,40,41}, this stroma remodeling occurs as early as 3 days post-bacterial administration and is sustained for at least 7 days without any aberrant adverse events, highlighting its specificity.

Moreover, through our immunotherapy combination studies, we have demonstrated that the administration of the programmable bacteria prior to ICI treatment potentiates the antitumor responses of immunotherapy, resulting in the regression of 4T1 tumors, whereas ICI monotherapy proved completely ineffective. This promising outcome was further corroborated by the survival study, revealing that one out of nine mice treated with ICI combination experienced complete remission, making the first study to provide evidence of the responsiveness of the 4T1 tumor model to immune checkpoint blockade. Likewise, we found that the bacteria significantly enhanced the antitumor efficacy in the E0771 tumor model. Specifically, mice bearing E0771 tumors exhibited a significant but modest response to ICI monotherapy, which was strongly augmented when mice were pre-treated with the bacteria. Importantly, primary tumor remission was evident in half of the mice treated with the ICI combination. Combining the bacteria with immunotherapy led to a complete cure for all mice of the E0771 study. Such survival benefit was also accompanied by the acquisition of immunological memory. In line with this, the engagement of HA with its binding receptor CD44, expressed on CD8 T cells, has been found to skew CD8 T cells toward a terminal effector differentiation state, reducing their ability to form memory cells. This suggests that HA degradation can impact the immune memory response⁴².

In our attempt to explain the differential response rates of the two triple-negative breast cancer models to ICI treatment, we investigated potential changes in the immune microenvironment of these tumors. Our findings indicate that in both 4T1 and E0771 tumor models, ICI alone or in combination reduced the levels of MDSCs. MDSCs mediate immunosuppression by depleting l-arginine, leading to T cell arrest ^43,44 and anergy ^45,46. Additionally, we observed a significant upregulation in the total macrophage population in E0771 tumors only after treatment with ICI combination, accompanied by an increase in the frequency of M1-like TAMs relative to the M2-like TAMs. M1-like TAMs promote antitumor immunity through direct cancer cell killing, secretion of pro-inflammatory cytokines such as interleukin-12 (IL-12), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ), and recruitment and activation of CD8⁺ T cells, DCs and natural killer (NK) cells ^47,48. Conversely, M2-like macrophages possess tumor-promoting capabilities involving immunosuppression, angiogenesis and tumor progression ^49,50.

Finally, we explored the applicability of our bacteria in treating CT26 colon adenocarcinoma as a model of stroma-poor tumors ⁵¹. C35R7-Hase bacteria treatment effectively reduced the HA levels but to a lesser extent compared to the 4T1 model, likely due to the lower levels of HA available for breakdown. The combination of the bacteria with ICI did not prove to be more beneficial compared to ICI monotherapy. Neither did we observe significant changes between ICI and combination treatment in the intratumoral immune cell infiltration and frequencies. Specifically, the same increase in the infiltration of helper and cytotoxic T cells was demonstrated in groups receiving the immunotherapy cocktail, compared to the control group, independently of the bacterial treatment. Assessment of the tissue stiffness and ECM content of untreated tumors revealed significant variation in stroma composition across the different tumor types employed in this study. CT26 tumors exhibited a 5-fold lower elastic modulus compared to 4T1 and E0771 tumors, confirming our hypothesis that enzymatic degradation of ECM can only benefit highly desmoplastic tumors. It further demonstrates the role of the ECM and particularly hyaluronan in inhibiting responses to ICI in solid tumors, as the CT26 tumor is shown to have less fibrotic stroma and also demonstrated higher baseline response levels to the ICI cocktail. Taken together, our study underscores the novelty and importance of stromal remodeling engineered live biotherapeutics as a promising therapeutic strategy for enhancing the efficacy of cancer treatment, with significant implications for future clinical applications.

Murine cell lines and cell culture

The CT26 murine colon carcinoma (CRL-2638) and 4T1 (CRL-2539) murine triple-negative breast cancer cell lines were obtained from ATCC while the E0771 (94A001) murine triple-negative breast cancer cell line was purchased from CH3 Biosystems. All cell lines were cultured in Roswell Park Memorial Institute medium (RPMI-1640, LM-R1637, biosera) supplemented with 10% fetal bovine serum (FBS, FB-1001H, biosera) and 1% antibiotics (A5955, Sigma). The CT26 cell line was bio-engineered to express firefly luciferase using lentivirus transduction. Briefly, virus production was performed by the co-transfection of 293T cells with the hygromycin-resistant bioluminescent plasmid (18782, Addgene) and the packaging plasmid pCL-Eco (12371, Addgene). For the in-house virus particle formation, 293 T cells were transfected with X-tremeGENE™ 9 DNA Transfection Reagent (Sigma), plasmids, gag, pol and env genes. After 24-48 h of incubation, virus particle-rich media was collected, centrifuged and passed through a 0.2 μm filter. CT26 cells were treated with the virus particles for 16 h. Then, the medium was replaced, and cells were cultured in a complete RPMI culture medium for a day. Finally, luciferase-transfected CT26 cells were cultured in a medium containing 750 µg/ml hygromycin B for 5 days and cloned until they reached confluency. Confirmation of luciferase expression was determined through bioluminescence imaging using confocal microscopy (Leica Stellaris 5). All cultures were maintained at 37 °C in a humidified incubator containing 5% CO2.

Bacterial strains and culture

E. coli strain was obtained from DSMZ (DSM 18039) and cultured in Luria Broth (Miller). The bacteria hyaluronidase (bH) (Q59801, UniProt) was codon optimised with a FLAG tag at the N-terminus and inserted into Neobe’s medium copy number expression vector using the previously published BASIC method⁵². Bacterial strains weretransformed by the heat shock method,cultured onto LB agar plates containing 100 μg/ml kanamycin and allowed to grow overnight at 37°C. Glycerol stocks were generated for positive clones identified by colony PCR and restriction digest of plasmid preparations. Just before the in vivo administration, glycerol stocks (in PBS buffer with 10% (v/v) glycerol) of different E. coli candidates with known concentrations were thawed and adjusted to the desired concentration (CFUs/ml) in PBS.

Drugs and reagents

The mouse monoclonal antibody anti-PD-1 (CD279, clone RMP1-14) and mouse monoclonal anti-CTLA-4 antibody (CD152, clone 9D9), and the InVivoPlus mouse IgG2b isotype control were purchased from BioXCell and dissolved in the recommended InVivoPure pH 7.0 Dilution Buffer (IP0070). The ICIs or the respective isotype controls were administered via intraperitoneal injection at a dose of 10 mg/kg for anti-PD-1 and 5 mg/kg for anti-CTLA-4 in line with previous studies ^32,53,54.

Dose studies

Six- to eight-week-old immunocompetent BALB/cOlaHsd female mice were used for the implantation of 4T1 cells. Breast tumors were established orthotopically by injecting 5 x 10⁴ cells under the mammary fat pad. Bacteria or PBS (phosphate-buffered saline, control group) were administered intravenously via a 50 μl injection at the desired concentration into the tail vein of tumor-bearing mice when the average tumor volume reached 150 mm³.

Six- to eight-week-old immunocompetent C57BL/6OlaHsd female and male mice were used to establish the CT26 tumor models. Firstly, 1 x 10⁶ cells were injected subcutaneously into the flank of mice to generate donors. When the tumors reached an average volume of 500 mm³, they were excised and cut into fragments of 1-2 mm³. The caecum of the recipient mice was exteriorized and slightly scraped using forceps. Tumor fragments were then implanted onto the caecum directly underneath the serosa to prevent the risk of bowel perforation. The caecum was returned to the abdomen and the peritoneum and skin were sutured. One tumor fragment per mouse was used in the implantation procedure ³³. Mice were allowed to recover from surgery for four days and then injected with the bacteria as described above.

Mice were euthanized 7 days post injection of bacteria. Tumors and spleens were removed and stored in culture media at -80°C until further processing. Planar dimensions (x, y) of 4T1 tumors were measured 2 to 3 times per week using a digital caliper, and tumor volume was estimated using the ellipsoid volume formula: π/6*D1*D2*D3, where D1 and D2 are the x, y dimensions and D3 corresponds to geometric mean of the planar dimensions x and y. Animal body weight was monitored before and after bacterial administration at frequent intervals. In total, 5-6 mice were employed per treatment group for both CT26 and 4T1 dose studies.

Immunotherapy combination studies

BALB/cOlaHsd and C57BL/6OlaHsd female mice aged between six and eight weeks were orthotopically injected with 5 x 10⁴ 4T1 and E0771 cells, respectively, under the mammary fat pad as described earlier. Once the tumors reached an average size of 150 mm³, mice were randomized according to tumor size into the following treatment groups: PBS (control group), C35R7 10⁶ CFUs (live biotherapeutic group), ICI (immunotherapy group) and C35R7 10⁶CFUs + ICI (combination therapy group). Mice received either a single i.v. injection of the C35R7 live biotherapeutic (10⁶ CFUs/mouse) or PBS and after three (for the 4T1 study) or four days (for the E0771 study), they received the first cycle of immunotherapy via an i.p. injection (10 mg/kg anti-PD-1 + 5 mg/kg anti-CTLA-4). In total mice were subjected to three cycles of ICI treatment, with a three-day interval between each. For both 4T1 and E0771 combination studies, the tumor dimensions were measured every 2 to 3 days with a caliper until their surgical resection 2 days post-last immunotherapy cycle. Tumors were collected and processed accordingly for the flow cytometry experiment, and the survival of mice was monitored daily.

For the CT26-luc tumor model, orthotopic tumors were established following engraftment of a tumor fragment onto the caecum of six- to eight-week-old immunocompetent BALB/cOlaHsd female and male mice as described before. Mice were randomized according to the bioluminescence signal on day 3 into the following treatment groups: PBS (control group), C35R7 10⁶ CFUs (live biotherapeutic group), ICI (immunotherapy group) and C35R7 10⁶CFUs + ICI (combination therapy group) and subjected to a single i.v. injection live biotherapeutic at a dose of 10⁶ CFUs on day 5 post-tumor implant. ICI cocktail was administered 3 days later and repeated for an additional 2 cycles with a three-day interval between each. Tumor growth was monitored every 2 to 3 days by bioluminescence imaging (BLI) using the AMI-HT (Spectral Instruments Imaging) imaging system until mice reached a humane endpoint. Fifteen minutes before in vivo imaging, tumor-bearing mice were intraperitoneally injected with D-Luciferin firefly (150 mg/kg in PBS, Biosynth) and anesthetized using 1–3% isoflurane. Regions of interest (ROIs) were drawn around the tumor region and the acquired signals were quantified as photons/sec with Aura Image software (version 2.8.5, XQuartz). The last recorded bioluminescence measurement of dead or sacrificed mice was considered for the calculation of mean tumor burden at subsequent time points.

Mice rechallenge with E0771

To investigate if mice presenting a complete response after drug treatment have also acquired immunological memory, we rechallenged the survivors with orthotopic inoculations of E0771 cancer cells (5x10⁴) 112 days after the initial cancer cell inoculation. As a control group we used 8 mice of the same age.

Ultrasound shear wave elastography and contrast-enhanced ultrasound

Assessment of tumor elastic modulus and perfusion was measured via shear wave elastography (SWE) and contrast-enhanced ultrasound (CEUS), respectively. For SWE, the Philips EPIQ Elite scanner with the eL18-4 linear array transducer was employed, according to previous research ⁵⁵. The method generates a shear wave velocity via an acoustic push pulse, creating a color-mapped elastogram (in kPa) where red indicates hard and blue soft tissue. A confidence display is also used as a reference for the shear wave quality of the user-defined region of interest (ROI). The average value of the tumor region is automatically generated by the system under default scanner settings and expressed in kPa. Two planar sections were collected from each tumor, and the average value of these sections was used to plot the data. The settings that were used were: frequency 10 MHz, power 52%, B-mode gain 22 dB, dynamic range 62 dB. SWE was performed at two different planes of each tumor and the average value of both planes was used for our analysis. For the 4T1 dose study, SWE was performed just before the administration of bacteria, at 3- and 7-days post-administration.

For CEUS, tumor perfusion was assessed after a bolus injection of 8 μl Sonovue/Lumason (in the USA) contrast agent (Bracco, Geneva, Switzerland). Sonovue consists of sulfur hexafluoride microbubbles encapsulated with a phospholipid shell and having a mean diameter of 2.5 μm. Before each ultrasound application, mice were anesthetized by i.p. injection of Avertin (200 mg/kg), and ultrasound gel was applied to the imaging region to prevent any pressure of the transducer on the underlying tissue. Ultrasound scanning of tumors was performed using the linear array transducer eL18-4. Contrast first harmonic signals were received at 8 MHz with a mechanical index of 0.06. For all subjects, the depth of the focus was set to 3 cm allowing measurements of the full depth of the tumor. The gain was set at 90% for each recording. Focus was optimized and standardized for each subject when finding the tumor area using B-mode imaging. Real-time power modulation imaging was initiated after flashing imaging with a high mechanical index to destroy the microbubbles in tumor tissue to peak contrast intensity to allow visualization of bubble replenishment. Image analysis was performed offline using quantification software (QLAB, Phillips Medical Systems). For the analysis, we plotted the linearized image intensity (after removal of the logarithmic compression) from a ROI around the tumor as a function of time (time-intensity curve, TIC). We also calculated the normalized perfused area when the image intensity reached its peak, as the number of pixels with microbubble signals divided by the total number of pixels of the tumor ROI. The probe was placed softly on the animal with excess ultrasound gel to avoid any pressure that could potentially alter the flow ^56,57.

Ex-vivo measurement of tissue elastic modulus

Ex-vivo assessment of the elastic modulus was determined using an unconfined compression experimental protocol as previously described ³⁴. Briefly, a small fragment of tumor (3 mm length x 3 mm width x 2 mm thickness) was placed at the base and compressed with a piston at the top side to 10% strain for 2 minutes to ensure that the piston applied well on the specimen and that the specimen's response to deformations was measurable. Next, the sample was compressed at 30% strain. The elastic modulus of the specimen was measured from the slope of the stress-strain curve between 20% and 30% compression.

Statistical analysis and reproducibility

Statistical analysis was performed using GraphPad Prism 9.0 software. For tumor growth curves and ultrasound measurements involving multiple time points, two-way analyses of variants (ANOVA) followed by Dunnett’s multiple comparisons were used. For other experiments including tumor mass measurements, toxicity assessment, histology and flow cytometry one-way ANOVA was used. Only statistically significant differences along with the exact P values are displayed in the figures. A P value of less than or equal to 0.05 was considered statistically significant. The sample size and repetition of each experiment are indicated in corresponding figure captions. Sample size was based on previously published studies measuring the same output variables. Data are presented as means with standard errors.

All in vivo experiments were conducted per the animal welfare regulations and guidelines of the European Union (European Directive 2010/63/EE and Cyprus Legislation for the protection and welfare of animals, Laws 1994–2013) under a license acquired and approved (CY/EXP/PR.L03/2020) by the Cyprus Veterinary Services Committee, the Cyprus national authority for monitoring the welfare of animals in research.

Data availability

All other data supporting the findings of this study are available from the corresponding author and the clinicians responsible for running the clinical study upon request. Additional methods employed in the study can be found in Supplementary Information.

Acknowledgments

This research was funded by the European Union (ERC project, LiveBioThx, 101100769). Views and opinions expressed are, however, those of the authors only and do not necessarily reflect those of the European Union or the European Research Council Executive Agency. Neither the European Union nor the granting authority can be held responsible for them.

Author contributions

All authors have reviewed and approved the manuscript to its final version. M.P. designed experiments, analyzed, and interpreted results and wrote the manuscript with contributions from all authors. F.M. performed in vivo treatments and ultrasound measurements. M.B.R. and O.M.T.P. performed in vitro experiments related to the various bacterial constructs and staining of tissue explants. C.V. helped with ultrasound measurements, analysis of immunofluorescence images, and statistical analysis. C.M. performed experiments related to tissue processing and immunostaining. P.C.S. and A.S., co-founders of Neobe Therapeutics Ltd, contributed to the design of the bacterial constructs and supervision of this work. T.S. designed experiments, supervised research, secured funding and was involved in project administration, writing, and editing.

Competing interests.

The authors declare no competing interests.

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Programmable bacteria act as live biotherapeutics to remodel tumor stroma and potentiate immunotherapy

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