The increasing amounts of diagnosed breast cancer have attracted public attention globally.^1-3 There are millions of people diagnosed with different subtypes of breast cancers, which represents the abnormal growth of epithelium cells of the ducts or lobules in the glandular tissue of the breast.^4-6 Some of them are non-invasive that are confined to the duct or lobule with no symptoms. Triple negative breast cancer (TNBC) is the most aggressive subtype with poor prognosis and high risk of recurrence. It can continuously progress and invade the surrounding breast tissues or even local metastasis to the nearby lymph nodes or distant metastasis to other organs.^7-9 Because of the lack of specific targets, the treatment of TNBC is highly challenging. We are aimed to develop an immune therapeutic approach that can convert the acidic and hypoxia microenvironment caused by the high glycolytic activity of breast tumor. Figure 1 represents the overview of our project below.

Figure 1. The overview of our project.

We are looking for an approach that can tackle with the acidic and hypoxia tumor microenvironment for efficient immunotherapy of triple negative breast cancer. Our effort is focused on the genetic engineering and chemical modification of E coli Nissle 1917 that can specifically attack on tumor cells and live on lactic acid. The consumption of lactic acid is expected to invert the acidic pH and re-boost macrophage-driven anti-tumor immune responses. Manganese dioxide nanoparticles are used as nano-immunomodulators that can catalyze hydrogen peroxide (H2O2) to produce oxygen (O2) under acidic conditions and promote innate and adaptive immunity.

Reprogramming of cellular metabolism is the emerging hallmark of TNBC that exacerbates proliferation, metastasis and angiogenesis of tumor cells.¹⁰The high glycolytic activity of TNBC cells usually causes the accumulation of metabolic byproduct lactic acid and hypoxia tumor microenvironment (TME).¹¹As shown in Figure 2, the acidification of tumor microenvironment can be sensed by tumor associated macrophages (TAMs) through proton-sensing G protein-coupled receptors (GPCRs).¹² The tumor acidosis subsequently induces the expression of a transcriptional repressor called ICER in tumor-associated macrophages, which are then converted from pro-inflammatory M1 phenotype into a non-inflammatory M2 phenotype.¹³ Classical M1 and alternative M2 activation of macrophages represent two dynamic changing states of macrophage activation.¹⁴In contrast to M1-type, cytokines released from M2-type macrophages promote but not inhibit the proliferation of contiguous cells.¹⁵ M1–M2 polarization of macrophage is usually tightly balanced by transcriptional and posttranscriptional regulatory networks.¹⁶ However, an imbalance of macrophage M1–M2 polarization often leads to immunoevasion including not only local metastasis to the nearby lymph nodes but also distant metastasis to other organs.¹⁷

Figure 2. Macrophage M1-M2 polarization induced by acidic tumor microenvironment (TME).

Triple negative breast cancer (TNBC) is the most dangerous subtype with least favorable outcomes.¹⁸ It is characterized by profound clinical features such as high invasiveness, high metastasis, high risk of recurrence, and poor prognosis.¹⁹ Because TNBC tumor does not express estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER-2), it lacks actionable targets for regular endocrine therapy or HER2 treatment.²⁰ Currently, the treatment of TNBC is still challenging. There is limited responses to chemotherapy. And surgical removal or radiation therapy are also not able to prevent the recurrence. Advances in omics technologies have revealed the dynamics of TNBC microenvironment heterogeneity.²¹ These new knowledges facilitate the development of novel poly (ADP-ribose) polymerase (PARP) inhibitors²² and antibody–drug conjugates.²³ In particular, immune-checkpoint inhibitors (ICI) have been revolutionizing the therapeutic opportunities for early-stage and advanced-stage TNBC patients.²⁴ Recent clinical trials have coupled paclitaxel with immune checkpoint inhibitors in TNBC patients.²⁵ Increased stromal tumor-infiltrating lymphocytes and micronucleation over baseline have been found.²⁶ It shows paclitaxel can induce chromosome missegregation on multipolar spindles during mitosis. Then post-mitotic cells containing micronuclei may activate cyclic GMP-AMP synthase (cGAS) and induce type I IFN responses in the stimulator of IFN genes (STING) pathway.²⁶ As shown in Figure 3, the combination of ICI with paclitaxel promotes the M1 polarization of TAMs and provides a new immunotherapy to treat metastatic TNBC.²⁷ However, while such microtubule-targeting agent potentiates an immune response in TNBC, the acquired resistance to ICI-based therapies has clinically emerged, which demands further development for the immunotherapy of TNBC.²⁸

Figure 3. Macrophage M1 polarization facilitated by the combination of immune checkpoint inhibitors with microtubule-targeting agent paclitaxel.

Our solution is aimed to rescue tumor associated macrophages (TAMs) and re-boost macrophage driven anti-tumor immune responses through the remodeling of the acidic and hypoxia tumor microenvironment. It is expected to convert TAMs from non-inflammatory back to inflammatory phenotype by adjusting the acidic pH and relieving hypoxia of TME. Because bacteria infection can induce innate and adaptive immune responses,²⁹ we hope combine the advantage of intrinsic antitumor activities of bacteria with nano-immunomodulators and enhanced capability to specifically attack on TNBC cells. E coli Nissle 1917 cells are genetically engineered with SIRPα gene that can recognize CD47 present on surfaces of TNBC cells.³⁰ Engineered bacteria then undergo surface thiolation through a simple one-step imidoester reaction by which primary amino groups on bacterial surface can be converted to free thiols under cytocompatible conditions.³¹ Gold nanoparticles are deposited on surfaces of hollow manganese dioxide nanospheres that are subsequently bound with thiolated bacteria because of the high affinity of Au nanoparticles with the thiol groups.³² The genetically and chemically modified live tumor-targeting bacteria are named as TuTaBa (兔大巴). As shown in Figure 4, TuTaBa is expected to live on lactic acid that is transported with monocarboxylate transporter 1 (MCT1) and not disturb the normal glucose metabolism. There are great advantages to use manganese dioxide nanospheres as nano-immunomodulators because they have shown lots of biological functions that can directly regulate tumor growth.³³ Cancer cells often produce large amounts of H₂O₂ and GSH for their metabolism and resistance to immunological killings.³⁴ With the peroxidase-like activity, MnO₂ nanospheres can effectively catalyze the in situ production of O₂ and relieve hypoxia by reacting with endogenous H₂O₂ to release Mn²⁺.³⁵ They can also react with GSH in tumor cells to generate GSSH and Mn²⁺.³⁶ The ability of MnO₂ nanospheres to modulate TME allows them to enhance the antitumor immunotherapy. When the mission is completed and the tumor microenvironment (TME) goes back to normal pH value, engineered bacteria undergo clearance program.

Figure 4. The sketch of genetically and chemically modified TuTaBa (tumor targeted bacteria).

Genetically or chemically modified bacteria have been recognized as new therapeutic tools that are able to grow in tumors and prevent their proliferation. The feasibility of bacteria-based treatment can be summarized as follows for the immunotherapy of triple negative breast cancer.

✓ Bacteria can initiate the antitumor effects by activating innate and adaptive immune responses or directly killing tumors with secreted toxins.²⁹

✓ With the aid of flagella, live bacteria with the facultative anaerobic property can naturally move toward solid tumors for survival and reproduction in which there are usually insufficient oxygen and low pH. High levels of local colonization of bacteria can be formed there.³⁷

✓ Bacteria can compete with tumors for nutrients that are required for cell metabolism. The colonization of bacteria in the surrounding areas of tumors reduces local nutrient supply. Tumor cells may then die from starvation and suffocation in resultant necrotic regions.³⁸

✓ Bacteria can live on lactic acid that is transported into cells by ubiquitously expressed MCT1. It is responsible for the transport of circulating lactate, pyruvate, and acetoacetic acid and also facilitates the unidirectional proton-linked transport of monocarboxylates across the plasma membrane. The research group supervised by Professor Bernhard Ø. Palsson has systematically investigated the adaptive evolution of the metabolic network of E. Coli strains on lactic acid.³⁹ They have experimentally demonstrated the convergence and reproducible growth of E. coli on lactic acid.⁴⁰

✓ Overexpression of cluster of differentiation 47 (CD47) has been found in triple negative breast cancer.⁴¹ Then it can be used as a marker for engineered bacteria to look for tumor cells. Meanwhile CD47 can also functions as the engulfment signal and dominant macrophage immune checkpoint that represent a signal “do not eat me” to the immune system.⁴² It has been demonstrated that the interaction of CD47 with its receptor signal-regulatory protein alpha (SIRPα) cause the suppression of phagocytic activities. The expression of SIRPα on bacteria should compete with the SIRPα on macrophage to interact with CD47 on TNBC tumor cells. Then the phagocytic and cytotoxic activities of macrophages against TNBC tumor cells would be enhanced.

✓There are increasing evidences that manganese can regulate immunological responses in different conditions.⁴³ Its role as an alarm protein of innate immunity and the regulation of adaptive immunity have been experimentally proved.⁴⁴ Under acidic conditions, MnO₂ nanospheres can efficiently catalyze the production of O₂ by interact with H2O2 to release Mn²⁺, or interact with the intracellular GSH to release Mn²⁺.⁴⁵

Compared with other types of invasive cancer, TNBC has fewer treatment options due to the absence of actionable targets. The proposed immunotherapy approach combines genetically engineered bacteria with hollow manganese dioxide nanospheres that function as chemical nano-immunomodulators. It not only remodels the acidic and hypoxia tumor microenvironment and re-boost macrophage-driven anti-tumor responses but also provide hollow nanospheres as potential carriers for the delivery of therapeutic payloads of clinical needs in the future. In particular, because the technique strategy is based on bacteria depletion of lactic acid instead of blocking or knocking out the generation pathway of lactic acid, it does not disturb normal glucose metabolism while the acidification of TME is stopped. This immunotherapeutic approach is helpful for those people suffering from chemotherapy and radiation that cannot prevent the recurrence of TNBC but may damage normal tissues. It is shown self-propelled bacteria can be developed as perfect robot therapies with genetic and chemical manipulation. The distinguished advantage is the capability to penetrate into solid tumor regions that are usually inaccessible to passive therapies. With the aid of the expression of genes that can recognize molecular markers on surfaces of tumor cells, differentiation of tumor cells with normal cells and specific target is possible. In the meanwhile, the chemical nano-immunomodulators relieve hypoxia and oxidative stress. Those unique features of genetically engineered and chemically modified bacteria make them well-suited as perspective anticancer agents.

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