Description

Engineered Bacteria for Immunotherapy of Triple Negative Breast Cancer



Introduction

Breast cancer refers to a disease in which lining cells (epithelium) of the ducts or lobules in the glandular tissue of the breast grow out of control.1-3 When the cancerous growth is confined to the duct or lobule, it is called in situ cancer that generally causes no symptoms. However, it may continuously progress and invade the surrounding breast tissues. Compared with other subtypes, triple negative breast cancer (TNBC) is most aggressive and associated with early metastatic recurrence, including local metastasis to the nearby lymph nodes or distant metastasis to other organs.4-8 The treatment of TNBC is still challenging due to limited chemotherapy responses and the absence of specific targets. Surgical removal, radiation therapy and chemotherapy are not able to prevent the recurrence. There are millions of women diagnosed with breast cancer globally according to the report of world health organization (WHO) in 2021.

Problem

The combination of immune checkpoint inhibition (ICI) with paclitaxel is emerging as a new immunotherapy to treat metastatic TNBC.9-11 However, the acquired resistance to ICI-based therapies is clinically emerging, which is a major challenge for immunotherapy in TNBC. The high glycolytic activity of TNBC tumors usually result in the accumulation of metabolic byproduct lactate and extracellular acidification.12-15 A family of proton-sensing G protein-coupled receptors (psGPCRs) can sense the acidic microenvironment including GPR4, GPR65/TDAG8, GPR68/OGR1 and GPR132/G2A.16, 17 When the acidic microenvironment is sensed by macrophages18, 19 and induces the expression of transcriptional repressor ICER,20, 21 tumor-associated macrophages are converted into a non-inflammatory phenotype (Figure 1).22-24 Such immunoevasion exacerbates proliferation, metastasis, and angiogenesis of tumor cells.25-27

Figure 1. Inflammatory and non-inflammatory phenotypes of tumor-associated macrophages

Goal

Our research goal is to rescue tumor associated macrophages (TAMs) and re-boost macrophage driven anti-tumor immune responses by converting tumor-associated macrophages from non-inflammatory back to inflammatory phenotype. Because bacteria infection can induce innate and adaptive immune responses,28 we hope combine the advantage of intrinsic antitumor activities of bacteria with enhanced features to treat triple negative breast cancer. Team efforts have been focused on the construction of Live tumor-targeting bacteria that are named as TuTaBa to specifically target breast cancer cells and clear away lactic acid in the microenvironment of glycolytic breast tumors (Figure 2). Engineered bacteria are expected to live on lactate so as not to disturb normal glucose metabolism. When the mission is completed, TuTaBa can undergo self-clearance through acid/base inducible promoters that are designed to stimulate the expression of functional genes .

Figure 2. The sketch of TuTaBa (tumor targeting bacteria)

Feasibility

Although bacteria are generally considered as pathogens that cause infections or even tumor development, the therapeutic role of bacteria has recently been recognized in medical and pharmaceutical fields.29 It has been realized that genetically modified and attenuated bacteria are able to grow in tumors and prevent their proliferation. The feasibilities of bacteria-based treatment of triple negative breast cancer are summarized as follows.

  • Bacteria infections induce innate and adaptive immune responses.
  • Bacteria have flagella that can make movement toward tumors. Secreted toxins and the formation of biofilms maybe helpful for the treatment of tumors.
  • Bacteria wrest the nutrients required for the metabolism of cancer cells. The entry and proliferation of bacteria in the surrounding areas of tumors reduce local oxygen and nutrient supply, leading to the formation of necrotic regions where tumor cells may eventually die from starvation and suffocation.
  • It is possible to engineer bacteria that live on lactate. The research group leaded by Professor Bernhard Ø. Palsson has systematically investigated the adaptive evolution of E. Coli strains on lactate by metabolic network responses.30 They observed replicate evolutionary experimental results that showed convergence and reproducible growth of E. coli on lactate.31 Techniques for the expression of monocarboxylate transporter (MCT) family specific for lactate has reported by Professor Patrick D. Bosshart group32 that should enhance the ability of engineered bacteria to live on lactate and clear the acidic microenvironment. These results provides us helpful information in engineering bacteria.
  • On TNBC tumor cells, surface proteoglycan chondroitin sulfate proteoglycan 4 (CSPG4) has been reported as a new target for the treatment of triple negative breast cancer. Professor Soldano Ferrone group has found that CSPG4 protein was preferentially expressed in 32 of the 44 (72.7%) primary TNBC lesions tested, in TNBC cell lines, and in tumor cells in pleural effusions from 12 metastatic breast cancer patients.33It is feasible to express monoclonal antibody scFv-Fc on engineered bacteria so that they can specifically targets to TNBC cells.
  • Cluster of differentiation 47 (CD47) is the dominant macrophage immune checkpoint that represents a signal “do not eat me” to the immune system. Several research groups including professor David S Sallman group have demonstrated that the interaction of CD47 with its receptor signal-regulatory protein alpha (SIRPα) cause the suppression of phagocytic activities.34 It is possible that the expression of CD47-targeting fusion protein SIRPαD1-Fc on bacteria should compete the interaction SIRPα on macrophage with CD47 on TNBC tumor cells and increases the phagocytic and cytotoxic activities of macrophages against TNBC tumor cells.
  • Acid/base responsible genes were reported in E. coli by several research groups such as Li group.35-39 The using of pH-sensing promoters is feasible to achieve the self-regulation of the expression of functional genes of engineered bacteria. When the mission is completed and the tumor microenvironment (TME) goes back to normal pH value, engineered bacteria initiate the expression of self-clearance genes.
Perspectives

Currently TNBC has fewer treatment options than other types of invasive cancers. Bacteria based immunotherapy of TNBC maybe helpful for those suffering from chemotherapy and radiation that are toxic to normal tissue and cannot completely destroy all cancer cells. With genetic manipulation, those self-propelled bacteria can be engineered as perfect robot therapies that can penetrate into tumor regions inaccessible to passive therapies, and specifically target to tumor cells. They can also be further enhanced as a versatile platform that can specifically deliver paclitaxel or other therapeutic payloads of clinical needs in the future. Although there are still many technical challenges, the unique capabilities of bacteria make them well-suited as perspective anticancer agents. All challenges should be addressed with advanced synthetic biological techniques.

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