What's the situation?
Breast cancer is the most common malignancy in women, and one of the three most common cancers worldwide, along with lung and colon cancer. In 2012, almost 1.7 million people were diagnosed worldwide and about half a million people died from this disease (2). Cure after cure has failed, and no treatment is foolproof. However, immunotherapy shows promise. the promise has been found to be curative in leukemia's, but not physical, solid tumors, such as a lump in a breast determining breast cancer. One of the reasons cancer is so hard to destroy is because the cancer cells will fool the human immune system.
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What are MDSCs?
Specifically, myeloid-derived suppressor cells (MDSCs) which suppress immunological responses by interfering with the effector functions of T cells, dendritic cells, and Natural Killer cells, can be manipulated into protecting the tumor. MDSCs suppress immunotherapies that is cart or adoptive transfer of T cells. MDSCs upregulate T cell regulation and suppress cytotoxic T cells. While the immune system would be able to destroy cancer, through macrophages and dendritic cells, the MDSCs suppress the natural response, meaning the immune system has type 1 immunity or cytotoxic T cells. Finding out why and how cancerous cells are manipulating MDSCs into protecting the tumor and fighting against treatments like chemotherapy and radiation could inform future cures, increasing efficacy and safety.
What do MDSCs do?
For many years, cancer research has been focused on blasting the cancerous tumor with radiation and chemicals, hoping to break it down over time and cause it to disappear completely. Clearly, this has not been working, and scientists have been focusing more and more on what is preventing the tumor from being destroyed. Myeloid-derived suppressor cells (MDSC) have been found as the perpetrator for these actions. MDSCs are a heterogeneous immature myeloid cell population that can suppress T-cell responses. These cells have been found in both humans and mice, as well as similar small rodents, which present an ideal model system to study the human response without testing on actual human subjects. These MDSCs studies have been completed in the immunoregulatory functions of the spleen as well as secondary lymphoid organs. The microenvironmental cues that drive the phenotype of MDSCs supporting tumors is hypoxia (a condition in which the body or a region of the body is deprived of adequate oxygen supply at the tissue level). While occupied in the hypoxia state, MDSCs are not able to work as applicable as need for the tumor or body, resulting in a hypoxia-responsive element (HRE) in the gene’s proximal promoter region.
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How do MDSCs impact immunotherapy?
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What is IL-22?
IL-22 is a cytokine that is hypothesized to be important for tumor progression. IL-22 plays an important role in tissue repair and inflammatory responses, and is implicated in the pathogenesis of psoriasis, ulcerative colitis, as well as liver and pancreas damage (1). The IL-22 gene is also hypothesized to be a recruiter for MDSCs, but it is not clear why this is possible.
T cells were first considered as the source of IL-22, and subsequent studies demonstrated that innate immune cells, including γδ T cells, NK T cells and innate lymphoid cells, can also secret IL-22 (1). This cytokine binds to a transmembrane receptor complex, spreading a signal to relevant cells in the body. Within the body, keratinocytes (KCs) are found in the skin and are the main type of Il-22 responsive cells. They are capable of producing and secreting various mediators of the inflammatory reaction and of the immune response. It has been demonstrated that the transcription regulation factors STAT1 and STAT3 control IL-22 expression in CD4+ T cells.
T cells were first considered as the source of IL-22, and subsequent studies demonstrated that innate immune cells, including γδ T cells, NK T cells and innate lymphoid cells, can also secret IL-22 (1). This cytokine binds to a transmembrane receptor complex, spreading a signal to relevant cells in the body. Within the body, keratinocytes (KCs) are found in the skin and are the main type of Il-22 responsive cells. They are capable of producing and secreting various mediators of the inflammatory reaction and of the immune response. It has been demonstrated that the transcription regulation factors STAT1 and STAT3 control IL-22 expression in CD4+ T cells.
What is CRISPR?
CRISPR (clustered regularly interspaced short palindromic repeats), which originates from bacteria as a proto-immune system, is used by the protein Cas9 (or "CRISPR-associated protein 9"), an enzyme that acts like a pair of molecular scissors, capable of cutting strands of DNA. CRISPR’s main use is changing DNA enough to suppress MDSC function. Ideally, one of the changed DNA sequences will result in the MDSC suppression themselves and allowing the immune system to fight the tumor.
CRISPR is used in many different types of scenarios, yet those over others are thoroughly used as a way for finding the effector proteins and their corresponding enzymatic activity, which is a prime note in the delineation of the current six major CRISPR- Cas system types. After being used enough, CRISPR gains adaptive immunity through three shared function stages acquisition of new spacers (adaptation), crRNA biogenesis (expression), and nucleic acid detection and destruction (interference). originally, CRISPR was discovered in bacteria acting as a functional adaptive immune system to fight off viruses. CRISPR is now used to target and cut specific sequences of DNA in cells other than bacteria. The CRISPR system provides prokaryotes with heritable immunity by obtaining foreign fragments of DNA and RNA, creating matching nucleic acid sequences and afterward destroying the viral template. CRISPR is not only used for DNA coding and manipulation but also genetic engineering applications in various organisms. The type of CRISPR-Cas system that targets DNA does this by scanning and interrogating the genomic DNA. CRISPR RNA spacers pair with the complementary segments in foreign DNA, altering the non-complementary strand to form an R-loop (an R-loop is a nucleic acid structure consisting of two antiparallel DNA strands plus one RNA strand). The type IIA Cas9 nuclease from Streptococcus pyogenes (SpCas9) is most widely used for genome editing. Not only does SpCas9 get used for DNA editing, but also type V Cas12a (or Cpf1) and type IIC Cas9 nucleases can be used in many applications, as they are extracted from various bacteria. Although this is a useful and intriguing tool, the molecular details of DNA interrogation and formation of an R-loop complex by CRISPR-Cas effectors are not fully understood, as CRISPR has only been used for genome editing for about 5 years.
CRISPR is used in many different types of scenarios, yet those over others are thoroughly used as a way for finding the effector proteins and their corresponding enzymatic activity, which is a prime note in the delineation of the current six major CRISPR- Cas system types. After being used enough, CRISPR gains adaptive immunity through three shared function stages acquisition of new spacers (adaptation), crRNA biogenesis (expression), and nucleic acid detection and destruction (interference). originally, CRISPR was discovered in bacteria acting as a functional adaptive immune system to fight off viruses. CRISPR is now used to target and cut specific sequences of DNA in cells other than bacteria. The CRISPR system provides prokaryotes with heritable immunity by obtaining foreign fragments of DNA and RNA, creating matching nucleic acid sequences and afterward destroying the viral template. CRISPR is not only used for DNA coding and manipulation but also genetic engineering applications in various organisms. The type of CRISPR-Cas system that targets DNA does this by scanning and interrogating the genomic DNA. CRISPR RNA spacers pair with the complementary segments in foreign DNA, altering the non-complementary strand to form an R-loop (an R-loop is a nucleic acid structure consisting of two antiparallel DNA strands plus one RNA strand). The type IIA Cas9 nuclease from Streptococcus pyogenes (SpCas9) is most widely used for genome editing. Not only does SpCas9 get used for DNA editing, but also type V Cas12a (or Cpf1) and type IIC Cas9 nucleases can be used in many applications, as they are extracted from various bacteria. Although this is a useful and intriguing tool, the molecular details of DNA interrogation and formation of an R-loop complex by CRISPR-Cas effectors are not fully understood, as CRISPR has only been used for genome editing for about 5 years.
What is the main use of CRISPR?
As the main focus for CRISPR has not always been for DNA editing, CRISPR is now the main use for experiments involving DNA replication or editing since approximately 4 years ago. The type IIA Cas9 nuclease from Streptococcus pyogenes (SpCas9) will be used through CRISPR to change genomes, and duplicate the DNA for cells to test if the IL-22 cytokine, through interactions with MDSCs, can affect the growth of cancer. The IL-22 gene is being tested as it is being knocked out 4T1 cell line, assessing changes in MDSC recruitment while this occurs. The cell cultures are created to test the productivity of these proteins, and how they may be utilized to research tumor development in the mouse model system. If the tumor has stopped growing or is shrinking, that is when success is discovered.
Moving to research
What is the purpose of this study?
In this research, tools such as CRISPR and flow cytometry will be used to analyze the actions and effects of IL-22 in 4T1 cancer cells taken from the mouse blood, spleen, tumor, tissue and bone marrow in balb/c mice. By knocking out the gene that encodes IL-22 and analyzing cell cultures, this project will explore the immune response and production of MDSCs with and without the cytokine. Transfection is the process with nucleic acids being introduced into eukaryotic cells. the DNA was taken and put into mammalian cells using lipo 3000. The goal is to ascertain the function and importance of the IL-22 gene to cancer’s manipulation of the immune system.
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