Identifying a novel mechanism that boosts the clearance of dead cells by macrophages

Cell death is an important process through which the structure of our bodies are shaped throughout development. For example, soft tissue cells between the fingers and toes undergo apoptosis (programmed cell death) to separate the digits from each other during development (Figure 1). Billions of cells die in our bodies every day and a prompt clearance of dead cells and their debris is important for maintaining tissue homeostasis. Tissue homeostasis requires a very tight control of the balance between cellular proliferation and differentiation.  The majority of these dead cells are cleared by macrophages, a type of immune cell,  through a process called “efferocytosis”. 

Figure 1: Programmed cell death is an important process during development that serves to remove superfluous cells and tissues. Figure was adopted from “Mechanical Regulation of Apoptosis in the Cardiovascular System”.

If dead cells are not appropriately cleared by macrophages, they start leaking material in the cellular environment that causes inflammation and tissue damage. Efficient efferocytosis prevents this from happening, and thereby protects tissues from inflammation. Macrophage-mediated efferocytosis is an important process to promote the resolution of inflammation and restore tissue homeostasis. While inflammation causes swelling, redness, and pain, efferocytosis does not. In fact, enhancing efferocytosis has the potential to dampen inflammation and reduce tissue necrosis which is caused by injury or failure of the blood supply. Defective efferocytosis contributes to a variety of chronic inflammatory diseases such as atherosclerotic cardiovascular disease, chronic lung diseases, and neurodegenerative diseases. Understanding the mechanisms that regulate efferocytosis could help us develop novel therapeutic strategies for diseases driven by defective efferocytosis and impaired inflammation resolution.

Like other cells in the body, macrophages need energy to maintain their activity. Glycolysis and oxidative phosphorylation are two major metabolic pathways to provide energy for cells. Glycolysis is a process in which glucose (sugar) is broken down through enzymatic reactions to produce energy. Macrophages take up glucose via glucose transporters on the cell surface, such as GLUT1. Glucose will be broken down to generate ATP (energy) and lactate, an end product of the glycolysis pathway.

A research group in the department of Medicine at Columbia University led by Dr. Maaike Schilperoort, a postdoctoral research scientist in Dr. Ira Tabas’ laboratory, identified a novel pathway in which efferocytosis promotes a transient increase in macrophage glycolysis via rapid activation of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2), a key enzyme in the glycolysis pathway to convert glucose to lactate (Figure 2). 

Figure 2: The engulfment of apoptotic cells by macrophages through efferocytosis increases glucose uptake via the membrane transporter GLUT1. Glucose is broken down into lactate through glycolysis, and this process is boosted by efferocytosis through activation of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2). Lactate subsequently increases cell surface expression of the efferocytosis receptors MerTK and LRP1. These efferocytosis receptors facilitate the  subsequent uptake and degradation of other apoptotic cells in the tissue. This figure was created using Biorender.

MerTK and LRP1 are so-called “efferocytosis receptors” that allow the macrophages to  bind to dead cells before they can engulf and degrade them. The current study found that the production of lactate leads to an increase in MerTK and LRP1 on the cell surface in a calcium-dependent manner to drive continual removal of dead cells (Figure 2). The authors mentioned that lactate promotes an efferocytosis-induced calcium-raising mechanism that could be involved in the mitochondria division. The mechanism of how lactate promotes the increasing of calcium is not well understood and needs to be explored more in the future. This finding provides potentially new therapeutic strategies for improving cell death clearance such as targeting an endogenous inhibitor of PFKFP2. This novel finding was published in Nature Metabolism in February 2023. 

Reviewed by: Maaike Schilperoort , Erin Cullen, and Sam Rossano

Engineered bacteria enhanced the current therapeutics in lung cancer

Lung cancer is the second most common type of cancer and is responsible for the most cancer-related death in the U.S. The American Cancer Society reports that more than 235,000 people were diagnosed with lung cancer in 2021. There are three major types of lung cancer: non-small cell lung cancer (85% of cases), small cell lung cancer (10% of cases), and lung carcinoid tumor (5% of cases). The causes of lung cancer include but are not limited to smoking, secondhand smoke, exposure to certain toxins, and family history. The symptoms include cough with blood, chest pain, wheezing, and weight loss when the cancer is in the advanced stage. Depending on the type of lung cancer and what stage it has progressed to, the treatment will be different. Broadly the treatment involves surgical resection, radiation, chemotherapy, targeted therapy and immunotherapy. However, to treat this complex disease researchers are always looking for new and improved treatment modalities.

A research group at Columbia Engineering led by Dr. Dhruba Deb in the lab of professor Tal Danino developed a new therapeutic to treat non-small cell lung cancer (NSCLC) by combining an engineered bacteria with targeted therapy to enhance the treatment efficacy without any additional toxicity in laboratory animal models. This finding was published in Scientific Report on December 13, 2022.

By engineering a toxin named theta (θ) toxin in the bacteria S.typhimurium and by testing the response of a variety of NSCLC cells to this engineered bacteria, the research group found that θ toxin can kill a variety of NSCLC cells even with different genetic background such as mutated growth factor receptor like KRAS or EGFR, the most common mutations found in NSCLC. The research group also administered locally live S.typhimurium expressing theta toxin (Stθ) in NSCLC tumor cells in the mouse model and found a 2.5-fold reduction of tumor growth within a week compared to the control group. 

With the success of testing live S.typhimurium expressing theta toxin (Stθ) in mouse model and no toxicity  found in the peripheral organs, the research group tested whether using the engineered bacteria could enhance the efficacy of the standard of care chemotherapies as well as small molecular inhibitors. To identify potential drugs to combine with Stθ, the authors used RNA-sequencing. This helped to pinpoint which biochemical pathways in NSCLC cells were helping the cells to survive the Stθ treatment. To overcome this ability of NSCLC cells, the researchers blocked those biochemical pathways with drugs and eliminated the NSCLC cells. For example, one of the drugs, MK2206 when paired with Stθ treatment, blocks the NSCLC cells’ ability to survive via biochemical signaling of phosphorylated AKT that promotes survival and growth in response to extracellular signals. Key proteins involved are PI3K (phosphatidylinositol 3-kinase) and Akt (protein kinase B)..  The research group also tested the combination treatment of MK2206 and Stθ in a mouse model. They found that the combination treatment of Stθ bacteria and MK2206 suppressed the tumor growth efficiently compared to treatment with only Stθ or only MK2206. Moreover, with lower dose of bacteria and drug use, they could observe similar treatment results and could possibly avoid  the activation of the immune system caused by high dose of bacteria treatment. Taken together, this combination treatment is a potential therapeutic for the NSCLC. 

There are several limitations in this study that need to be addressed before entering the clinical trial. First, the authors used a small number of animals per cohort in the in vivo study, so they plan to expand their study to assess the overall survival upon treatment. Second, the toxins themselves are not selectively targeted to the cancer cells, so they need to develop a selective delivery method to avoid the systemic toxicity. In the laboratory animal models, the local administration of the live bacteria acted as a selective delivery. But, further studies are necessary to use this live bacteria in human clinical trials. Overall, this study opens up new treatment options for patients diagnosed with the NSCLC.

Reviewed by: Sam Rossano, Margarita T. Angelova

A high-tech device to effectively deliver drugs to tumors in the brain

Brain and other nervous system cancer is the 10th leading cause of death for men and women. Around 18,280 adults died from primary brain and central nervous system tumors in the United States in 2020. Glioblastoma is the most common malignant brain and other CNS tumors and median survival is only 12-15 months.

Why is brain tumor hard to treat? It is due to the blood brain barrier (BBB), a specialized network of blood vessels and cells that shields and protects the central nervous system against circulating toxins or pathogens that could cause brain infections. However, the impenetrability of the BBB also makes it difficult to treat tumors in the brain compared  to those in other organs. Patients with brain tumors have to receive higher doses of chemotherapy to penetrate the BBB and ensure an adequate amount of medication reaches into the brain and kills the tumor cells. The higher dose of chemotherapy will lead to the toxicity to the normal cells, which can result in serious side effects and even death of the patient. To overcome the BBB, scientists have tried to develop many different methods to deliver drugs effectively to the brain so that lower doses of chemotherapy can be used.

Over the last decade, Drs. Bruce and Canoll’s laboratory at the Columbia University Medical Center has been developing a new method to directly administer drugs to the site of the brain tumor, which they call convection enhanced delivery (CED). In CED, a small pump is implanted into the abdomen and connected to a thin catheter under the skin. Wireless technology is used to turn the pump on and off and control the flow rate of medicine that seeps in the tumor tissue.

In a recent study with the CED device, Dr. Bruce used topotecan, a drug that is toxic to glioblastoma cells, to treat five patients who were at least 18 years old with recurrent brain tumors. The patients were infused with topotecan for 48 hours, followed by a 5–7 day washout period before the next infusion, with four total infusions. Patients went about their normal routines at home while treatment continued without any severe side effects. After the fourth infusion, the pump was removed and the tumor was resected. This method is in the early phase of clinical trials (phase 1b) and will be expanded to a larger scale of patients due to test the safety and efficacy of the therapy for recurrent glioblastoma. This novel chemotherapy delivery strategy overcomes the limitation of drug delivery in patients with glioma. The results from this study have recently been published in Lancet Oncology 

There are two limitations in this study. First, there is no comparison group for determination of definitive survival benefit. Second, there is no way to assess the disease progression and treatment response due to effects of local drug infusion and surgical resection. However, in the locally delivered therapy (CED method), the authors used patients as their own control by performing pre-therapy and post-therapy MRIs and PET scans. The CED device effectively gets through the BBB to kill the brain tumor so new classes of drugs and targeted compounds could potentially be used such as high-molecular-weight compounds or viruses.

Reviewed by: Pei-Yin Shih, Maaike Schilperoort

Lactic acid – a new energy fuel source in brain tumor

What does lactic acid do to the body?

Lactic acid is produced when the body breaks down carbohydrates in low oxygen levels to generate energy. It is mainly found in muscle cells and red blood cells. An example of lactic production is when we perform intense exercise. 

Glucose, glutamine, fatty acids, and amino acids are well-known energy sources for cell growth and division. In the past, lactic acid has been known as a by-product of glycolysis, a process in which glucose is broken down through several enzyme reactions without the involvement of oxygen. However, recent studies showed that lactic acid is a key player in cancer cells to regulate tumor cell growth and division, blood vessel formation, and invasion. The tumor cells prefer to use glycolysis to produce energy and lactic acid despite the abundance of oxygen levels. Lactic acid is an alternative fuel source for glucose-deprived tumors to avoid cell death.

Lactic acid is transported through the membrane via the monocarboxylate transporter 1 (MCT1). A research group at Columbia University led by Dr. Markus Siegelin in the department of Pathology and Cell Biology showed a substantial presence of lactic acid in the citric acid cycle (TCA cycle), a series of chemical reactions to generate energy, in the glioblastoma cells cultured in the nutrient deprivation condition (low glucose and glutamine concentration). When the glucose and/or glutamine concentrations increased, less lactic acid was involved in the TCA-cycle metabolites. The uptaken lactic acid in the TCA-cycle was traced by using a method called C13 carbon tracing and was analyzed by liquid chromatography-mass spectrometry to identify the structure of different molecules. The researchers concluded that lactic acid is used as a fuel source to generate the energy in the brain tumor cells. Furthermore, lactic acid is converted to Actetyl-CoA and contributed to the gene modification in glioblastoma cells (Figure 1). These novel findings were published in a prestigious journal,  Molecular Cell

Figure 1: Role of lactic acid in the epigenetic modification of glioblastoma cells. Lactic acid is transported to the membrane via the monocarboxylate transporter 1 (MCT1) and contributed to the TCA cycle as a fuel source to generate the energy. Lactic acid is converted to Actetyl-CoA and contributed to the gene modification in glioblastoma cells. Suppressing the TCA cycle by using the targeted drug, namely CPI-613 (devimistat) leads to the abrogation of lactic acid in the energy production. The figure was generated by Biorender.

From these findings, the authors proposed to use CPI-613 (devimistat) drug, which targets TCA-cycle metabolites (Figure 1), to  treat glioblastoma cells. Indeed, CPI-613 showed a suppression of cellular viability in vitro of glioblastoma cells and an extension of the animal survival curve in the mouse model. The authors suggested that the combination of CPI-613 with other standard care treatment in glioblastoma such as temozolomide and radiation could be a potential clinical therapy for patients with glioblastoma.

Read more about this exciting finding here:

https://www.sciencedirect.com/science/article/pii/S1097276522006475 

Reviewed by: Pei-Yin Shih, Sam Rossano, Emily Hokett

Metastatic cancer cells have unstable DNA which helps them to evade the body’s immune system

Melanoma brain metastasis (MBM) frequently occurs in patients with late stages of melanoma (skin cancer). It is the third leading cause of brain metastases after lung and breast cancers. Cancer cells break away from the primary tumor and travel to the brain through the bloodstream. Despite significant therapeutic advances in the treatment of metastatic cancers, MBM  remains a challenging problem for therapeutic treatment due to the blood brain barrier. The MBM may develop a variety of symptoms that are similar to primary brain tumors such as headache, difficulty walking, or seizures. To provide comprehensive studies of the cells inside melanoma brain metastases, Jana Biermann, a postdoc in Dr. Benjamin Izar’s lab at Columbia University, performed single-cell-sequencing, nucleus RNA-sequencing, and CT scans of 22 treatment-naive MBM and 10 extracranial melanoma metastases that could spur the development of a new generation of therapies (Figure 1).

Figure 1: A comprehensive study of melanoma brain metastasis and extracranial melanoma metastases by performing single-cell genetic analyses of frozen brain samples. snRNA-seq: single nuclei RNA sequencing; TCR-seq: T cells sequencing. Image was created from BioRender based on Figure 1A of the original article that was published in CellPress with title “Dissecting the treatment-naive ecosystem of human melanoma brain metastasis”.

The authors also analyzed the genes expressed in 17 melanoma brain metastases and 10 extracranial melanoma metastases patients. The data revealed unstable DNA in the melanoma brain metastases compared with extracranial melanoma metastases. The unstable DNA triggers signaling pathways that enable the tumor cells to spread around the body and to suppress the body’s natural immune response that normally fights off the tumor cells. The researchers also found that the relocated melanoma cells adopt a neuronal-like state that might help tumor cells adapt and survive after they migrate to the brain. Furthermore, by using CT scans of multiple slices of the tumors, researchers created three-dimensional images of the tumors and uncovered heterogeneity in metabolic and immune pathways within and between tumors. 

The authors also found that the cancer cells in the brain significantly expressed  several genes that are known to promote cancer progression, such as MET and PI3K, while the extracranial melanoma metastases strongly expressed genes related to epithelial cells, which are the cells that cover the inside and outside of the surfaces of your body such as skin and blood vessels. Understanding these pathways will help for the therapeutic targets. 

A limitation of the study is that the authors did not compare melanoma brain metastasis and extracranial melanoma metastases within the same patients, which could have introduced variability in their dataset. Nevertheless, the atlas that they built provides a foundation for further mechanistic studies on how different perturbations could influence brain metastasis ecosystems.

Reviewed by: Pei-Yin Shih, Sam Rossano, Maaike Schilperoort

Why do COVID-19 patients have trouble breathing?

The COVID-19 pandemic has resulted in over 145 million positive cases and 3.1 million deaths globally (32 million and 570,000 in the USA, respectively), as reported on April 26, 2021. Approximately 15% of infected patients with SARS-CoV-2 die from respiratory failure, making it the leading cause of death in COVID-19 patients.

A research group at Columbia University led by Dr. Benjamin Izar identified substantial alterations in cellular composition, transcriptional cell states, and cell-to-cell interactions in the lungs of COVID19 patients. These findings were published in the prestigious journal Nature. The team performed single-nucleus RNA sequencing, which is a method for profiling gene expression in cells, of the lungs of 19 patients who died of COVID-19 and underwent rapid autopsy. The control group included seven control patients who underwent lung resection or biopsy in the pre-COVID-19 era (Figure 1).

Figure 1: An overview of the study design wherein single-nucleus RNA sequencing was used to characterize lungs of patients who died from COVID-19-related respiratory failure. A) The lung tissue was extracted for mRNA, a genetic sequencing of a gene. B) The mRNA sequence will be read by a computer system. C) The gene expression of cells in the lung of COVID-19 patients samples and control samples. PMI: post-mortem interval. snRNA-seq: single nucleus RNA sequencing. QC: quality control.

The lungs from individuals with COVID-19 were highly inflamed but had impaired T cell responses. The single-nucleus RNA sequencing showed significant differences in cell fractions between COVID-19 and control lungs both globally and within the immune and non-immune compartments. There was a reduction in the epithelial cell compartment, which are the surfaces of organs in the body and function as a protective barrier. There was also an increase in monocytes (i.e., white blood cells that are important for the adaptive immunity process) and macrophages (i.e., cells involved in the detection, phagocytosis and destruction of bacteria and other harmful organisms), and a decrease in fibroblasts (i.e., cells that contribute to the formation of connective tissue) and neuronal cells. These observations were independent of donor sex. 

Monocyte/macrophage and epithelial cells were unique features of a SARS-CoV-2 infection compared to other viral and bacterial causes of pneumonia. The reduction in the epithelial cell compartment was due to the loss of both alveolar type II and type I cells. Alveolar type II cells repopulate the epithelium after injury, and provide important components of the innate immune system. Alveolar type II cells adopted an inflammation-associated transient progenitor cell state and failed to undergo full transition into alveolar type I cells, resulting in impaired lung regeneration. 

Myeloid cells (i.e., monocytes, macrophages, and dendritic cells) represented a major cellular constituent in COVID-19 lungs and were more prevalent as compared to control lungs. The authors found that the receptor tyrosine kinase that is important for coordinated clearance of dying/dead cells and subsequent anti-inflammatory regulation during tissue regeneration was downregulated. These data suggest that myeloid cells are a major source of dysregulated inflammation in COVID-19.

The authors also found significantly more fibroblasts in COVID-19 lungs than in control lungs. The degree of fibrosis correlated with disease duration, indicating that lung fibrosis increases over time in COVID-19. 

In this article, the authors mentioned the limitation of the study that they  analyzed lung tissues from patients who died of COVID-19, and therefore they only examined a subset of potential disease phenotypes. Based on the author’s observation, the rapid development of pulmonary fibrosis is likely to be relevant for patients who survive from severe COVID-19. This atlas may inform our understanding of long-term complications of COVID-19 survivors and provide an important resource for therapeutic development.

Read more about this article here: A molecular single-cell lung atlas of lethal COVID-19

Reviewed by: Molly Scott and Maaike Schilperoort

Survival of the fittest – how brain tumor cells adapt their metabolism to resist treatment

Glioblastoma WHO grade IV (GBM) is the most common primary brain tumor in adults. The therapeutic options for this recalcitrant malignancy are very limited with no durable response. A recent research article published in Nature Communications identified how the tumor cells alternate their metabolism to survive using targeted drug treatment in cell lines and mouse models. The project was led by Dr. Nguyen, a postdoctoral research scientist in Dr. Siegelin’s lab at Columbia University. 

Most cancer cells produce energy in a less efficient process called “aerobic glycolysis”, consisting of high levels of glucose uptake and generate lactic acid in the cytosol in the presence of abundant oxygen. This classic type of metabolic change provides substrates required for cancer cell proliferation and division, which is involved in tumor growth, metastatic progression and long-term survival. Dr. Siegelin’s laboratory at the Department of Pathology and Cell Biology at Columbia University Medical Center focuses on targeting cell metabolism and the epigenome for brain tumor therapy by using clinical validated drugs to suppress the tumor growth in glioblastoma. In this study, the authors used Alisertib (MLN8237), a clinically validated highly specific Aurora A inhibitor to target brain tumors. Aurora A kinases (AURKAs) are important for the proliferation and growth of solid tumors, including glioblastomas. Here, the authors found that Aurora A simultaneously interacts with both c-Myc (MYC Proto-Oncogene) and GSK3β (Glycogen Synthase Kinase 3 Beta). AURKA stabilizes the c-Myc protein and promotes cell growth. AURKA inhibitor, displayed substantial downregulation of the c-Myc protein. c-Myc (MYC) is an oncogenic transcription factor that facilitates tumor proliferation in part through the regulation of metabolism. Inhibition of Aurora A will lead to a degradation of c-Myc mediated by GSK3β. The authors also found that inhibition of Aurora kinase A suppressed the glycolysis signaling pathway in glioblastoma cells which was related to the degradation of c-Myc protein (Figure 1).

Figure 1: In the cells, Aurora A binds to c-Myc and facilitates cell proliferation. Inhibition of Aurora A will stop the cell from generating energy through glycolysis, a metabolic pathway that converts glucose to energy in cytosol, due to the degradation of c-Myc. c-Myc is marked for degradation by its phosphorylation at position T58 mediated by GSK3β. To survive, the cells start to use different pathways to generate energy by e.g. burning fat or proteins. Figures created with Biorender.com.

In addition to the acute treatment, it is important to understand how tumor cells acquire mechanisms to escape from chemotherapy following constant exposure to a drug and identify means to prevent this phenomenon from occurring. The research group generated drug-resistant cells by culturing them in the presence of alisertib for two weeks. These cells acquire partial resistance to alisertib and display a hyper-oxidative phenotype with an increase in the size of mitochondria with a tubulated shape. 

The chronic Aurora A inhibited cells were analyzed for the expression of genes that were modified after constantly applying the same dose of alisertib for a long term period. Researchers found that  resistance alisertib cells activate oxidative metabolism and fatty acid oxidation such as an increase in the generation of fatty acid proteins. These observations prompted them to test the hypothesis that alisertib along with fatty acid oxidation inhibitors such as etomoxir will reduce the cellular viability of glioblastoma cells. Etomoxir is a clinically validated drug that binds and blocks the mitochondrial fatty acid transporter. The authors found that the combination treatment of alisertib and etomoxir resulted in enhanced cell death as compared to single treatments and vehicles.

Given the significant promise of in vitro studies, the researchers extended their study in vivo by injecting the patient-derived glioblastoma cells acquired from the patient brain tumors in immunocompromised mice. Such model systems are currently considered to be in closest resemblance to the patient scenario. They found that the combination treatment extended animal survival significantly longer as compared to single treatment with alisertib or etomoxir, suggesting potential clinical efficacy. Taken together, these data suggest that simultaneous targeting of oxidative metabolism and Aurora A inhibition might be a potential novel therapy against deadliest cancers.

Article reviewed by: Maaike Schilperoort, Vikas Malik, Molly Scott, Pei-Yin Shih and Samantha.

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