Cancer+and+the+Immune+System

=About Our Cancer Project = ===Our project aims demonstrate how macrophages enhance tumor progression and identify effective immunotherapy strategies for combatting tumor associated macrophages ===

The Hallmarks of Cancer by Hanahan and Weinberg explained the importance of the immune system in the development of the correct "tumor microenvironment" for cancer to survive and thrive. Specifically how cancer develops the ability to evade the immune system, and promote inflammation.These two characteristics were identified as an "emerging hallmark" and "enabling characteristic", respectively. This indicated the importance of immune system manipulation to cancer survival. Further research into the role of cancer immunotherapy indicated that as soon as the right receptors signal the immune system to cancer cells, the cancer cells are subsequently completely destroyed by the immune system. This again reinforced Hanahan and Weinberg's emerging hallmark: "avoiding immune destruction" as a characteristic vital to cancer growth and survival, because of the volatility of cancer growth when the immune system can sense the presence of the cancerous cells.

Outline
1. Introduction to the Immune System 2. Strategies of Tumor Evasion 3. Immune System Enhancing tumor cell growth 4. Macrophages and Tumor Enhancement 5. Inflammation: Central to TAM Mediated Tumor Progression 6. MHC class IIlow and MHC class IIhi TAMS and Tumor Enhancement 7. Application to Immunotherapy 8. Conclusions

=**1. Introduction to the Immune System **= We will be covering only the basics of the immune system, particularly the components we will cover in later sections (Macrophage Enhanced Tumor Progression).

**Lymphocytes **
Lymphocytes is a sub set of the white blood cells of the Immune System, there are many different types of Lymphocytes, but those that are pertinent to this project are: B cells, Helper T cells, Cytotoxic T cells, and Natural Killer Cells.

i) B Cells

 * B cells ** mature in bone marrow and make up about 5-15% of the lymphocytes. They develop into plasma cells which secrete antibodies.

**ii) T Cells**
There are three major types of **T cells: ** **helper T cells ** that that secrete cytokines and help other immune cells such as the B cells and macrophages; **regulatory T cells ** suppress the immune response of antigens; **cytotoxic T cells ** destroy tumor cells and infected cells.

iii) Natural Killer Cells
Natural Killer cells are part of the innate immune system and thus serve as non-specific destroyers of pathogens. They are considered to be "the first responders" of the immune system and play an important role in tumor immune evasion.

**Major Histocompatability Complex Molecules **
** Major Histocompatability Complex (MHC) **molecules are cell surface proteins that a responsible for aiding in luekocyte (white blood cell) interactions as well as marking cell tissue types (vital for organ transplant tissue matching). Here we will focus on it's luekocyte interactions as it pertains to the immune system. Antigen presentation is their most important role, the basically provide the immune system with the ability to find out what is happening within our cells from extracellular surface. MHC proteins will detect proteins within the cell and express them as antigens on the cell surface, allowing the immune system to differentiate between self antigens vs. foreign antigens (1).

There are three classes of MHC molecules, but the first two classes are what apply to our Cancer Project: I and II. **MHC I** proteins are found on all extracellular membrane surfaces and they present synthesized proteins on their cell surface. Killer T cells will in turn use their MHC receptors to identify normal vs. abnormal body cells. **MHC II** proteins are found on the surface immune cells and they present only specific peptide antigens which are designed send signals to helper T cells to stop the body from attacking its own cells (1).

**Antigen Presenting Cells **

 * <span style="font-family: Arial,sans-serif; font-size: 10pt;">Dendritic cells **<span style="font-family: Arial,sans-serif; font-size: 10pt;"> prevalent in the skin attract antigens and present them to T cells while those in follicles bind with immune complexes and present those complexes to B cells. They are thought to be the major presenting cells and have the ability to induce T- and B-cell responses.

<span style="font-family: Arial,sans-serif; font-size: 10pt;">Among the phagocytes, which adhere to and ingest foreign bodies, are **<span style="font-family: Arial,sans-serif; font-size: 10pt;">monocytes **<span style="font-family: Arial,sans-serif; font-size: 10pt;">. These are made in the bone marrow and released into the blood system, where they mature into **myeloid cells**. Most important to our discussion are the **<span style="font-family: Arial,sans-serif; font-size: 10pt;">macrophages **<span style="font-family: Arial,sans-serif; font-size: 10pt;">. Out of the many characteristics of macrophages is the ability to release cytokines which "increase the expression of adhesion molecules on endothelial cells, facilitating leukocyte [white blood cell] influx and destruction of pathogens" ( <span style="color: blue; font-family: Arial,sans-serif; font-size: 10pt;">3 <span style="font-family: Arial,sans-serif; font-size: 10pt;">). They may also engulf and digest the pathogens (**phagocytosis**) or mark the infected cells to alert other immune cells which then identify and respond to the invader. Macrophages are able to wander around the blood vessels and can migrate into cell tissue through a process called **<span style="font-family: Arial,sans-serif; font-size: 10pt;">extravasation **<span style="font-family: Arial,sans-serif; font-size: 10pt;"> ( <span style="color: blue; font-family: Arial,sans-serif; font-size: 10pt;">4 <span style="font-family: Arial,sans-serif; font-size: 10pt;">). Ironically, it is because of these abilities and the fave that they can be manipulated that macrophages are recruited to tumor sites. Macrophages that are involved with tumor cell progression are referred to as **tumor-associated macrophages (TAM).**

<span style="font-family: Arial,sans-serif; font-size: 10pt;">In an article by Craig Murdoch and other researchers, other myeloid cells have been found to be associated with tumor cells. These include the following: **<span style="font-family: Arial,sans-serif; font-size: 10pt;">TIE2-expressing monoctyes (TEM) ** which express the angiogenic receptor TIE2 and can be utilized by tumor cells in angiogenesis; **hemangiocytes** which are a mixture of immature myeloid cells with pro-angiogenic properties; **myeloid-derived suppressor cells** which are also made up of myeloid cells, have the ability to suppress T cell and natural killer cell functions, and can change their shape in order to blend in with endothelial cells; **neutrophils**, leukocytes involved in inflammatory response; and **mast cells**, which mostly work in allergic reactions and autoimmune responses <span style="font-family: Arial,sans-serif; font-size: 10pt;">.

<span style="font-family: Arial,sans-serif; font-size: 10pt;">Returning to the topic of macrophages, there are several subpopulations of macrophages, as identified by Jeffrey Pollard and Bin-zhi Qian in accordance to immunological responses. They note that "because of their potential diversity of gene products, blood origin, and motile nature, they are ideally suited to perform specific tasks in a timely and spatially appropriate manner ... distinct tasks appear to require subtypes of macrophages" (5). **Activated** macrophages, like the name implies, are primarily involved in the T cell response to pathogens. They have "elevated expression of major histocompatibility complex (MHC), expression of interleukin, and tumor necrosis factor α" (5). They are also involved in making reactive oxygen and nitric oxide, and thus have the ability to kill pathogens. **Alternatively activated** macrophages are involved in humoral immunity and would healing. **Antigen-presenting** macrophage, like dendritic cells, will alert the rest of the immune system to the pathogens, as mentioned above. Of course, there are still other macrophages that are not necessarily actively involved in immunity. .

There are many different classes of **antigens** that can be found to be expressed in antigen presenting cells. As we know antigens are The author of [|this article], breaks antigens into six categories: (i) mutation specific antigens (p53, BCR-ABL, RAS, and CDK4) (ii) tumor specific rearrangements of of the heavy-chain locus (idiotype of B cell neoplasms) (iii) differentiation antigens (tyrosinekinase, MART-1, mucin) (iv) viral antigens (EBNA1, HPV E6/E7) (v) cancer antigens (CT, also found in testes) (vi) amplified or over expressed antigens in tumors (WT, MDM2, HER-2/neu)

The same article also identifies two types of antigens which are specific to cancer. **Tumor Specific Antigens** are antigens which are found on cancerous cells only and serve as specific signals for cancer cell destruction. As it would be expected, the mutations which are associated with theses antigens are tumor suppressor and oncogenes. These mutations are specific to cancer survival and thus are isolated to cancer cells. **Tumor Associated Antigens** are antigens which are found to be associated with both cancerous and noncancerous cells, they also call for the destruction of the cancer cells.

=2. Strategies of Tumor Evasion= According to Seliger's article, "Strategies of Tumor Immune Evasion" she identifies how a cancer cell signals for it's self destruction. Antigen presenting cells are the first step to this process. Once an antigen is expressed by a cell it will activate the T cell receptor/CD4/CD8 complex on the cell's membrane and this will lead to a signaling cascades occur which results in the activation of the transcriptional activation of genes responsible for the differentiation and the proliferation of T cells. T cells then proceed to lyse the antigen presenting cells. It is very important to note that there is a distinction between antigen presenting cells in different literature which is confusing if not understood correctly. As discussed under "Major Histocompatibility Complex Molecules"

T cells are not the only products of the immune system that cause this destruction Natural killer cells and cytotoxic T lymphocytes do as well. They follow the same lytic process as above, but use different specialized receptors (6). Generally, the methods of tumor immune evasion with the loss of antigens is to effect the T cells. The two ways that this happens is through either preventing T cells from "picking up" the cancerous cell foreign antigen signals by effecting antigens, or, the direct destruction of T cells. In this project we will be focusing on the signaling changes that occur inside and outside a cancer cell. For more information about cancer cell destruction of immune system components refer to the wiki "__ **How Cancer Eliminates Normal Immune Responses** ____ **"** __

The suppression of antigen expression in a cell is one of the most effective and common ways to avoid immune system detection. Antigen expression allows for the destruction of the antigen presenting cell by T cells as described in detail under "The Immune System" above. Many cancer cells have been found to have major alteration in the MHC I and II antigen processing. Specifically these mutations are often the downregulation of MHC I molecules on the tumor's surface. This downregulation is achieved with point mutations and inhibition of promoter activity. Interestingly enough, Seliger says that Natural Killer cells can still detect these cancer cells rather efficiently and can lead to tumor elimination (6). However, there are specific alterations that do cause cancer cells to be "invisible" to the immune system; antigen loss and the disruption of proteosome subunits.

=3. Immune System Enhancing Tumor Cell Growth= Tumor cells are able to change their microenvironment in order to influence the role and function of certain immune cells. For instance, macrophages are versatile and can "[alter] their phenotype to suit the microenvironment in which they reside" (8). Upon first observation, these immune cells which normally defend the host against tumor cells are like double edged swords. Myeloid cells, including macrophages, neutrophils, mast cells, eosinophils, and dendritic cells, "have an important role in regulating the formation and maintenance of blood vessels in tumors," according to a review by Craig Murdoch et. al. Although we will be focusing on enhancement by macrophages in this wiki, we will first explore some of the myeloid cell types and their relation to tumor progression.

> > > > > > > > In this article titled, "Traitorous Immune Cells Promote Sudden Ovarian Cancer Progression," The Wistar Institute details a study on the effect of dendritic cells on ovarian cancer. Past case studies show that an aggressive form of ovarian cancer may start off as malignant (due to supression suppression from the immune system) and then suddenly turn into metastatic cancer without warning. According to the Tumor Microenvironment and Metastasis Program of Wistar’s Cancer Center, the dendritic cells of the immune system might be the cause of the tumor "escaping" the immune system. > > Scientists working in the program at have created a model to work on understanding and preventing the growth of such tumors. Jose Conjeo-Carcia, one of the lead scientists said, “You can see where, if one ovary is cancerous, it is almost unrecognizable until an instantaneous moment, when it explodes into exponential growth. The key to this moment, our evidence suggests, is in the phenotypic changes taking place in the dendritic cells that are part of the tumor microenvironment” (18). > > Conejo-Garcia continued to say, "Interestingly, the tumors themselves are still immunogenic—they could still otherwise elicit an immune response—it is just that the dendritic cells are actively suppressing the involvement of other anti-tumor immune cells; primarily T cells” (18). So instead of necessarily promoting growth of tumor cells, dendritic cells have an inhibitory effect on the activity of those cells that are meant to help fight off the tumor cells. The findings of the TMMP team show that the dendritic cells actually suppress the activity of the T-cells, so the problem is in the immune system not being able to fight off the tumor cells efficiently after some point in the struggle. Conejo-Garcia and the rest of the team have since been looking into a way to win back dendritic cells after they defect.
 * **<span style="font-family: Arial,sans-serif; font-size: 10pt;">TIE2-expressing monoctyes (TEM): **<span style="font-family: Arial,sans-serif; font-size: 10pt;">These cells are identified by their expression of TIE2, an angiogenic receptor. The exact mechanism through which TEM is recruited to tumor sites is still unclear, but it is inferred that tumors selectively recruit TEM because, compared to other tumor-associated immune cells, they are “a more potent source of pro-angiogenic signals (8). Because TEM are found near tumor vasculature, “these cells might contribute to the regulation of tumor angiogenesis” (8). In an experiment done on mice, the ablation of TEM in tumors showed a reduction in angiogenesis and growth.
 * **<span style="font-family: Arial,sans-serif; font-size: 10pt;">Hemangiocytes: ** Hemangiocytes are "a heterogenuous mixture of immature and differentiated myeloid cells that express both [cytokines] VEGFR1 and CXCL12 ... have potent pro-angiogenic properties in vivo" <span style="font-family: Arial,sans-serif; font-size: 10pt;">(8) . As far as function, these cells work to repair and restore tissue perfusions. Both cytokines can transport these cells out of the bone marrow and help direct their movement. VEGFR1 hematopoietic progenitor cells have angiogenic properties and are "thought to help stabilize blood vessels through the release of angiogenic factors" <span style="font-family: Arial,sans-serif; font-size: 10pt;">(8) . When the levels of VEGFR1 and CXCL12 are high, hemangiocytes are recruited to hypoxic areas and promote angiogenesis of the tumor cells.
 * **<span style="font-family: Arial,sans-serif; font-size: 10pt;">Myeloid-derived suppressor cells (MDSC): **MDSC, like hemagiocytes, are a mixture of cells including immature myeloids (predecessors of neutrophils), monocytes, and dendritic cells. These cells can suppress the anti-tumor functions of other immune cells like T cells and natural killer cells. MDSC are found to be in high levels in the bone marrow of tumor-bearing mice (and humans). After being recruited to tumors, MDSC can also increase the growth rate of tumor cells and in angiogenesis <span style="font-family: Arial,sans-serif; font-size: 10pt;">(8 <span style="font-family: Arial,sans-serif;">) . It is noted that "cell-cell contact between MDSC and tumor-associated macrophages (TAM) resulted in ... a tumor-promoting type 2 response facilitating tumor progression" <span style="font-family: Arial,sans-serif; font-size: 10pt;">(8 <span style="font-family: Arial,sans-serif;">) . Along with this, MDSC are able to change their shape and blend in with endothelial-like cells, an ability that may contribute to tumor angiogenesis.
 * **<span style="font-family: Arial,sans-serif; font-size: 10pt;">Neutrophils: **These are a subpopulation of leukocytes that are primarily involved in inflammatory responses against invaders. High levels of neutrophils have been shown to correlate with poor prognosis. When CXCL8, secreted by tumor cells is in abundance, neutrophils are recruited to those sites <span style="font-family: Arial,sans-serif; font-size: 10pt;">(8 <span style="font-family: Arial,sans-serif;">) . These cells also promote neo-vascularization. The tumor-associated neutrophils are a source of MMP9, which is involved in the breakdown of the extracellular matrix. These neutrophils can thus promote angiogenesis through the production of this enzyme. Another function of neutrophils is their ability o secrete "a number of soluble pro-angiogenic factors that influence tumor angiogenesis ... [this] stimulates neutrophil de-granulation, releasing VEGF from intracellular stores, which subsequently induces endothelial cell proliferation" <span style="font-family: Arial,sans-serif; font-size: 10pt;">(8 <span style="font-family: Arial,sans-serif;">) . The release of these factors work in conjunction with factors released by the tumor cells in what seems to be a positive feedback loop.
 * **Eosinophils:** For now, more research is needed to reach a deeper understanding on how eosinophils can influence tumor progression. These cells are usually involved in response to parasitic infections and allergic reactions. Studies show that "eosinophils recruited to tuor sites an influence angiogenesis. Freshly isolated human blood eosinophils induce endothelial cell proliferation in vitro and angiogenesis, suggesting that eosinophils can directly influence angiogenesis" (8). In addition, eosinophils are able to secrete pro-angiogenic factors.
 * **<span style="font-family: Arial,sans-serif; font-size: 10pt;">Mast cells (MC): ** These cells are mostly involved in allergic responses and in "generating and maintaining innate and adaptive immune responses as well as in the development of autoimmune disorders and tolerance" (8). High numbers of MC have been observed around tumors before angiogenesis. They can also release angiogenic cytokines and have angiogenic proteases in their secretory granules, so it is thought that MC are recruited to tumor sites and release those factors to direct angiogenesis.
 * **<span style="font-family: Arial,sans-serif; font-size: 10pt;">Dendritic cells (DC): **<span style="font-family: Arial,sans-serif; font-size: 10pt;">There are two subpopulations of dendritic cells identified by Craig Murdoch et. al : myeloid DC (MDC) and plasmacytoid DC (PDC). MDC "have high phagocytic capabilities and, once they process foreign antigen, they become activated, undergo maturation and migrate to lymphoid tissue where they initiate activation of antigen-specific T cells" (8). Tumor cells have a high abundance of immature DC, which promote tumor angiogenesis better than their mature counterparts. Immature DC release pro-angiogenic cytokines and produce other factors which secrete angiogenic growth factors.

=4. Macrophages and Tumor Enhancement=

Introduction to Macrophages
Macrophages can be seperated into two general categories: M1 and M2 macrophages. M2 macrophages suppress inflammation and facilitate wound healing by promoting angiogenesis and tissue remodeling, and are therefore associated with tumorogenesis because of their ability to promote cell growth with the expression of various mitogens. M1 macrophages are associated with anti tumor responses. In "normal" immune conditions they are responsible for the signaling of immune cells responsible for cell destruction to an inflammatory site.



According to a study done by John Condeelis and Jeffrey Pollard published in 2006, macrophages are "at the center of an invasion microenvironment." They note a strong correlation "between macrophage density and poor patient prognosis ... there is a strong association between poor survival and increased macrophage density in thyroid, lung, and hepatocellular cancers" (9). These immune cells facilitate tumor cell angiogenesis, extraceulluar matrix breakdown, and also promote tumor cell motility. Their research shows that communication between macrophages and tumor cells allows the latter to invade and navigate the blood stream (intravasation). These tumor cells require some support from the stroma in order to fully grow. In the stroma are fibroblasts, adipocytes and blood - all of which exhibit the ability to enhance tumor progression to some degree. In a more recent report by Pollard and Bin-Zhi Qian, they state that “defined subpopulations of macrophages are responsible for these tumor-promoting activities.”

During invasion, hematopoietic cells, which include the tumor-associated macrophages, are also recruited to tumor sites. Condeelis and Pollard believe that there are two important findings that shed light on the the role of macrophages in cancer progression: 1. There is an association of chronic inflammation with the onset of cancer; furthermore, they believe that treatment with anti-inflammatory drugs reduces cancer risk. 2. "A high density of these tumor-associated macrophages correlates with poor prognosis in over 80% of studies published" (10)

The phenotype (several macrophage phenotypes are discussed in the Immune System section above) of macrophages during initiationn is the activated type. After tumor cells progress towards malignancy, however, "the macrophage phenotype changes from the 'inflammatory' type to one that resembles macrophages that promote tissue formation during development" (5). When the host undergoes inflammation, macrophages can act as the first signal response cells. They can find the antigens and mark them for the rest of the immune defense response. This process involves matrix remodeling, epithelial migrationm and angiogenesis. Tumor cells can then recruit macrophages and create microenvironments by causing them to alter their roles to those found in repair, and making use of "their matrix remodeling capacities, synthesis of growth and angiogenesis factors, and their engulfment of apoptotic cells" (10).

Six Important Traits of Macrophages
Condeelis and Pollard also identified six traits of macrophages that enhance tumor incidence, progression and metastasis: chronic inflammation, matrix remodeling, tumor cell invasion, intravasation, angiogenesis, and seeding at distant sites.

> > >
 * **Inflammation**: Inflammation seems to be the root cause of many types of cancer. In a mouse model of lung cancer, researchers have found that "bronchial exposure with //Haemophilus influenzae// lysate results in inflammation in the lung and an increase in tumorigenesis" (5). Furthermore, experiments have shown that the use of antibiotics to suppress bacteria reduces inflammation and inhibits tumorigenesis. Immune cells are always recruited to sites of inflammation, especially in cases of chronic inflammation. This type of inflammation that causes cancer is termed **smoldering inflammation.**
 * **Matrix Remodeling**: Macrophages are usually found in association with blood vessels. Researchers found that tumor cells tend to migrate towards blood vessels, and it seems that the two seem to migrate together or towards each other. Tumor cells take advantage of the matrix remodeling abilities of macrophages, which allow them to pass through the surrounding stroma and through the borders of basal lamina (10).
 * **Tumor cell invasion & Intravasation**: Macrophages can act as the keys that unlock a gate, allowing tumor cells to escape into the host's system. Tumor cell invasion and migration seems to occur sporadically and "tumor cell movement in vivo occurs adjacent to macrophages in the PyMT mammary tumor model" (11).

> Intravasation plays a big role in tumor progression as it is through this process that tumor cells are able to navigate through the blood stream of the host organism. We believed that with macrophage recruitment and utlizing them for intravasation and invasion, tumor cells are then able to metastasize using the other traits of macrophages. Using macrophages' matrix degrading capabilities, cancer cells can cross into the basal lamina of normal tissues and "infect" them. Tumor cells are able to recruit more macrophages through CSF-1 signaling. These macrophages and tumor cells then seem to gradually move towards the blood vessels together. > > CSF-1, or macrophage colony-stimulating growth factor, is a cytokine growth factor that is normally produced by osteoblasts and leads to the stimulation of the proliferation, differentiation, and survival of macrophages (14). It has been found that CSF-1 is overexpressed in many cancers such as breast, ovarian, endometrial,, and hepatocellular, and colorectal cancers. This overexpression has been linked to recruitment of macrophages to the tumor microenvironment leading to poor patient prognosis due to an accelerated rate in tumor invasion and metastasis (5). > > As shown in Figure #4 above, the data gathered suggest the possibility of a paracrine signaling loop between the tumor cells and macrophages. Tumor cells secrete CSF-1, which attracts macrophages and induces them to express EGF. Carcinoma cells exhibit movement towards blood vessels as a result of chemotaxis, which respond to EGF expression. Inhibition of either EGF or CSF-1 signaling pathways will result in the inhibition of migration of both. Furthermore, CSF-1 is regulated by steoid hormone receptor coactivator-1 (SRC-1), and without SRC-1, macrophages will no longer be recruited. This, tumor cell growth and metastasis are halted as well. > > Although CSF-1 plays an important role in macrophage recruitment, there are other factors which work with it. CCL22, is a gene that codes for the transcription of proteins that bind to the chemokine receptor CCR4 and plays a role in the signaling of T cells to inflammatory sites, however the exact method of which this is carried out is not well known (15). Cervical cancer patients have shown that the absence of this gene improves overall patient survival. This is an interesting result indicating that we do not know the full transcriptional function of this gene (5). > > Upon removal of macrophages from mice (by targetting the CSF-1 macrophage growth factor), the rate of tumor progression greatly reduced. Thus, this shows a "causal relationship between poor prognosis in a variety of reproductive tumors and overexpression of CSF-1, which recruits macrophages" (10). Overexpression of the CSF-1 growth factor is associated with poor prognosis in different tyes of cancers. Along with that, the chemokine CCL-2 is found to be overexpressed in some cancers; "its absence is associated with increasesed survival in cervical cancer patients" (5). Treatment through "antibodies that inhibit CSF-1 or its receptor reducses macrophage recruitment ... thereby inhibiting tumor growth and metastasis" (5). As with many processes, there are always exceptions. Macrophages in bone marrow sites phagocytose cells that don't express a certain antideath receptor - CD47. In luekemia, tumor cells will upregulate CD47 to escape death. Liver macrophages, which are also known as Kupferr cells) can also engulf surrounding tumor cells. The depletion of these Kupferr cells normall makes things worse and enhances metastasis. In this case, the tumor cells can grow faster in the absence of macrophages > . > Intravasation occurs through the help of clusters of macrophages located near blood vessels (11). Macrophages seem to give off recruiting signals with which tumor cells can use to migrate towards the vessels. Data shows that "reduction in the number of tumor-associated macrophages ... reduces the numbers of circulating tumor cells ... the paracrine loop between the two cell stpes is required for egress into the circulation in vivo" (5). In addition, in order for migration to occur, the ECM must be destroyed to allow the tumor cells to escape the basement membranes from primary tumor sites. > > > According to Qian and Pollard, there is a "dramatic enhancement of vascular density from the benign-to-malignant transition, a process referred to as the **angiogenic switch**" (5). Studies have shown that macrophages play a big role in the angiogenic switch and are even required. Overexpression of CSF-1 causes a premature accumulation of macrophages, which results in an early angiogenic switch. This allows tumor cells to quickly become malignant. Futher studies show that injecting tumor cells with CD11b-positive myeloid cells (markers of mature endothelial cells) will enhance angiogenesis. > Hypoxia is another big factor that contributes to angiogenesis. Macrophages pile up in hypoxic areas of tumors, where HIF1α regulates the transcription of genes like VEGF that turn on angiogenesis. Overexpression of VEGF in mice with no macrophages has been found to increase vascularization while speeding up the change to malignancy (12).
 * **Angiogenesis**: Studies show a correlation between the amount of local macrophages and areas where angiogenesis occur. It seems that the inhibition of MMP9, a metalloproteinase found in macrophages, blocks the release of VEGF, and therefore inhibits angiogenesis. Thus, “these data strongly suggest that macrophages play a role in both the initiation of angiogenesis in avascular areas and in the remodeling of the vasculature once formed to give coherent vascular flow" (10).

> > Circulating tumor cells can exit blood vessels and then “establish a proliferative niche where angiogenesis is necessary for sustained growth" (9). Agressive tumors can also stimulate the growth of smaller areas of metastasis using blood cells. These tumor cells express osteopontin, which is required for the mobilization of these bone marrow cells. "Primary tumors also cause the accumulation of myeloid derived cells at distant sites, and this process enhances metastatic efficiency" (5). These sites are called **premetastatic niches**. Myeloid chemoattractants are required for formation of the premetastatic niche. Myeloid cells reqruited to these sites secrete MMP9, which releases VEGF, which increases metastatic efficiency. Without VEGFR1, the premetastatic niche will not form. These niches act as sites to which tumor cells can adhere and grow. Some researchers suggest that the niches can also prepare the tissue for colonization. > > Interestingly, metastatic cells can grow in some tissues without primary tumors. In order to maintain growth, macrophages are recruited to these sites. Studies show that removal of these macrophages will reduce extravasation and metastatic seeding efficiency.
 * **Seeding at distant sites**: For metastasis to occur, the tumor cells must first be released from the primary sites and then transported through the ciculations or lumphatics in order to proceed to a distant site. Tumor cells can release thousands of cells every day, but only a few are successful at metastasizing (most of them die because of improper extravasation). When primary tumors release cells, they also secrete other factors to help them seed at distant sites. The tumor-derived extracullular matrix protein versican is one helper. As tested in the Lewis lung carcinoma, versican promotes metastasis through TLR2 signaling in myeloid cells (5).

Macrophages normally are able to bring antigens to T cells, which in turn kill them. In the tumor microenvironment, macrophage and other immune responses are suppressed. These macrophages' phenotypes change to one that will promote tumor growth. Macrophages in tumor areas grow and develop quickly due to high concentrations of CSF-1 secreted by tumor cells. Though they normally work in conjunction with T cells, they can inhibit the responses of cytotoxic T cells in several different ways. One mechanism involves the production of IL-10, which causes monocytes to express a programmed death ligand and suppresses T cell responses.Macrophages can also make the chemokine CCL22, which regulates regulatory T cells that actually suppress the cytotoxic T cells. Having many of the regulatory T cells may also lead to poor survival

In conclusion, Condeelis and Pollard suggest that macrophages make excellent targets for small molecule drugs as they "do not harbor malignant mutations and therefore have a stable genome" and are not likely to develop drug resistance.

=**5.** Inflammation: Central to TAM Mediated Tumor Progression=

Hanahan and Weinberg highlight the importance of inflammation in the tumor micro environment and identified it as an "emerging hallmark". While conducting our research, we decided that the most important trait that macrophages contribute to the tumor environment is inflammation. This conclusion was formed based on the following:
 * Macrophages play a critical role in the invasiveness of a tumor by the six traits discussed above. Without the addition of macrophages in the tumor environment, tumors would not metastasize at an increased rate, and a tumor would have a much higher chance at remaining benign (17,21)
 * Inflammation is the factor that calls pro-tumor M2 macrophages to the tumor micro environment initially, and it the primary signaling pathway, along with CSF-1 that continues to call these macrophages thereby enhancing inflammation (5,22)
 * The macrophage inflammatory phenotype retains the ability to promote angiogenesis and metastasis without switching to an alternate specific promoting phenotype (5)

The transcriptional factors NF<span style="font-family: Calibri,sans-serif; font-size: 11pt;">κ B and STAT3 also work as negative controls for each other. NF<span style="font-family: Calibri,sans-serif; font-size: 11pt;">κB causes inflammation, and is seen as "essential for the inflammatory phenotype" (5). Inhibition of the this signal will reduce inflammation, thus stopping tumor progression. Qian and Pollard conclude that "the immune system is normally in balance, but that once the negative controls of the immune responses are compromised, a persistent inflammatory response to normally commensal organisms results. This inflammation in turn creates a tumor-promoting microenvironment" (5).

NF<span style="font-family: Calibri,sans-serif; font-size: 11pt;">κ B is essential to tumor inflammation. It an be expressed by both the tumor cells and TAMs. NF<span style="font-family: Calibri,sans-serif; font-size: 11pt;">κ B is considered to be a "non-classical oncogene" this is because its activation rarely occurs as a result of mutation, rather it depends on signaling by neighboring cells. This signaling can be initially activated by many factors, some examples are(22) :


 * 1) Tobacco smoke
 * 2) Deprived blood supply --> Necrosis of tumor cells ---> Inflammation
 * 3) Cancer therapy such as radiation and chemotherapy

Once NF<span style="font-family: Calibri,sans-serif; font-size: 11pt;">κ B is activated a feedforward loop occurs between it and Tumor Necrosis Factor alpha (TNFalpha) and this then takes the inflammation to the next level. TNFalpha is expressed only by macrophages and is vital for not only the promotion of new inflammation but the promotion of angiogenesis and metastasis. This again describes the vitality of this pathway for tumor invasiveness.



Above in Figure #5 the feed forward loop is illustrated NF<span style="font-family: Calibri,sans-serif; font-size: 11pt;">κ B activates TNFalpha through IKK, also important inflammation promoting interluekins are activated as well.



In Figure #6 above, the role of TNF-alpha is illustrated. Note that it plays a role in Proliferation, Metastasis, and Angiogenesis. This shows the importance of not only the inflammation pathway but the amount of damage that can be done by the activation of TNF-alpha alone.

=**6.** MHC class IIlow and MHC class IIhi TAMS and Tumor Enhancement=

Introduction
While clinical studies have shown that macrophages promote tumerogenesis, there have been a good amount of studies that show that macrophages inhibit tumerogenesis as well. What seperates these "good" vs. "bad' macrophages are transcriptional signatures associated with each. This idea of "good" vs. "bad" macrophages caught our attention and so we looked at the following paper which analyzed and identified two types of tumor associated macrophages and seperated the two in terms of promoting tumerogenesis.

In a paper published in 2011, Benfan Wang and other researchers took a closer look at the role of macrophage subsets in tumor growth and progression. While macrophages account for many of the immune cell population in tumor sites, they are a heterogeneous population with different subsets responsible for different roles. Wang et al. identified two tumor-associated macrophage (TAM) subsets "based on their expression of MHC class II molecules, namely MHC class IIlow and MHC class IIhi <span style="font-family: Calibri,sans-serif; font-size: 11pt;">(17). They used clondronate-encapsulated liposomes in their experiments as they "cause irreversible damage to macrophages in vivo and decrease the number of infiltrating macrophages" <span style="font-family: Calibri,sans-serif; font-size: 11pt;">(17). With Cl2MDP-liposomes, they were able to see the role of TAMs through partial deletions.

All figures below have been taken from this article written by Benfan Wang et al. We kept the figure numbers as they were and analyzed the researchers' methods for their experiments and the conclusions drawn from their results.

Figure 3
In Figure 3, Wang et al. looked at the expression of TAM-associated suppressive factors, IL-10, TGF-β, VEGF, and MMP-9. <span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">As shown in Figure 3A, expression of all four factors was significantly decreased in cells treated with Cl 2 MDP-<span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">liposomes compared to the control. As shown in Figure 3B, they also looked at expression, which is found on the surface of macrophages and other monocytes. They found that “tumor infiltration by TAMs plays an important role in tumor progression” (17). Cl 2 MDP-<span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">liposomes decrease the amount of macrophages, which then causes a decrease in all the factors above. This then halts tumor growth and also decreases microvessel density, as shown in Figure 3C.

Figure 4


<span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">Next, they isolated TAMs to divide them into subgroups using f luorescence-activated cell sorting (FACS)<span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">. They found two populations of TAMs – MHC class II low and MHC class II hi TAMs, shown in Figure 4A. They found that each subset of TAMs display a set of factors in varying levels. MHC class II low TAMs expressed high levels of YM1, MGL1/2, Fizz1, Arg-1, msf, IL-10, TGF- β, MMP-9, VEGF, and PTGE2. MHC class II hi expressed high levels of IL-1β, IL-6, IL-12, and INOS. These are all shown in Figure 4B. They checked the expression of IL-10 in both subsets through staining experiments. They found that IL-10 expression in the MHC class II low TAMs was 42% higher 10 days after inoculation while the MHC class II hi TAMs only expressed at a 18% proportion (Figure 4C - not shown) (17). In As shown in Figure 4D (not shown), they analyzed the two subpopulations every 7 days for 4 weeks and found that an increase in MHC class II low TAMs correlated with a decrease in T cell proliferation. In Figure 4E (not shown), they showed that the number of MHC class II low TAMs “markedly increased as the tumor progressed, indicating that infiltrating macrophages preferentially differentiate into MHC class II low TAMs as tumors continue to grow” (17). They then looked over the expression of the subsets in untreated and TAM-depleted tumors. As reported in Figure 4F, they found that there were many more MHC class II low TAMs in the treated tumors. Meanwhile, the number of MHC class II hi TAMs was unaffected by Cl 2 MDP-<span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">liposomes.

<span style="font-family: Arial,sans-serif;">Because the MHC class IIlow and llhi TAMs express different levels of different growth factors, transcription factors and cytokines, Wang et al. seemed to classify them based on this expression. What is shown in Figure 4F is that there is increased hi and low expression. However, this paper suggests that CL2 inhibits tumor growth, and that both MHC class IIhi and IIlow should be at a lower expression. The conclusion states that low TAMs are alternatively activated and promote tumor growth. Cl2 should be decreasing TAM expression and therefore suppressing tumor growth. Figure G also shows an increased MHC class IIlow expression in the Cl2-treated cells compared to the PBS control, whereas figure H shows a decrease of MHC class IIlow TAMs in Cl2-treated cells. In the report, however, Wang et al. conclude that population of the MHC class II hi TAMs was unaffected by Cl 2 MDP-<span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">liposomes. This figure shows a difference, although not a huge one compared to the difference between the two samples of MHC class IIlow TAMs. Still, we do not believe that the researchers can say that the MHC class IIhi TAMs were completely unaffected by the presence of the Cl2MDP liposomes.

Figure 6
In order to delve into the effect of T cell activation on tumor progression, Wang et al. then looked at the rol each TAM subsets plays in activating T cells. In Figure 6A, they plotted the ratios between TAMs and T cells for both subpopulations, concluding that the MHC class IIhi TAMs promoted T cell proliferation while MHC class IIlow TAMs suppressed T cell proliferation (Figure 6B). Using antibodies to block IL-10 and TGF-β activity, they were also able to see that T cell proliferation was inhibited in the absence of these blocking antibodies. In Figure 6C, they showed that the reverse was true as well by measuring IL-10 and TGF-β in the supernatants of TAMs. From this, they concluded that MHC class IIlow TAMs “contribute to tumor progression via IL-10/TGF-β-mediated suppression of T cell activation” (17). They then looked at the other two important factors, MMP-9 and VEGF. In Figures 6D and 6E, they showed that the expression of both these factors was lower in MHC class IIhi TAMs than in MHC class IIlow TAMs. Wang et al. also ran an invasion assay to determine the effects of the subsets on the invasiveness and metastasis of tumor cells. As Figure 6F and 6G show, the MHC class IIlow TAMs increased the invasiveness of tumor cells.

Our issue with this figure was that the researchers did not note the discrepancies in the ratios shown in Figure 6A. Although the bars are clearly not level, they have chosen to draw the conclusion that MHC class IIhi TAMs promoted T cell proliferation. The OD values go start at ~0.4 and then jump past 0.9, then the T cell proliferation starts to decrease. In figure 6B, Wang et al. only show the T cell proliferation in regards to MHC class IIlow TAMs, which seems to be insufficient for drawing this conclusion that proliferation decreased as a result of their presence. Because IL-10 was found to be in greater proportions in these TAMs as compared to MHC class IIhi TAMs, it seems redundant to use them in their experiments as they were not testing for the effects of IL-10 and TGF-β.

Discussion
In conclusion, Wang et al. showed that Cl 2 MDP-liposomes depleted the population of macrophages around tumor sites without affecting the tumor growth itself. Their experiments showed that the "Cl 2 MDP-liposomes mediated inhibition of tumor growth is positively correlated with a decrease in TAM infiltration ... increased TAM infiltration contributes to the formation of a suppressive tumor environment and the promotion of tumor growth." (17). In addition, MHC class II<span style="font-family: Calibri,sans-serif; font-size: 8pt; vertical-align: super;">hi TAMs are more prevalent in the early stages of tumor growth and are suppressive of tumor growth. In contrast, the MHC class I I <span style="font-family: Calibri,sans-serif; font-size: 8pt;">low subset of TAMs activated and promoted growth. A transition between the two classes "is closely related to tumor progression, during which the predominant MHC class II<span style="font-family: Calibri,sans-serif; font-size: 8pt; vertical-align: super;">hi subset may shift to an MHC class I I <span style="font-family: Calibri,sans-serif; font-size: 8pt;">low subset" (17). When the number of MHC class I I <span style="font-family: Calibri,sans-serif; font-size: 8pt;">low TAMS was high, T-cell proliferation was inhibited, and it is through this inhibition that these TAMs are able to contribute to tumor growth and progression. Wang et al. concluded, "tumor progression is positively correlated with the number of infiltrating MHC class I I <span style="font-family: Calibri,sans-serif; font-size: 8pt;">low TAMS but negatively correlated with the number of infiltrating MHC class II<span style="font-family: Calibri,sans-serif; font-size: 8pt; vertical-align: super;">hi TAMs" (17).

=7. Application to Immunotherapy=

There are many different ways by which Immunotherapy can be used to combat cancer. Some methods involve triggering the signaling of immune system components such as T cells, natural killer cells, and cytotoxic lymphocytes to cancer cells to be lysed and destroyed. (6) This focuses more on cancer cell techniques for Immune Evasion. Methods involving Macrophages were found to be easily divided into two strategies: Destroying and "Re-educating".

Destroying TAMS
In an articlepublished in 2006, <span style="font-family: Arial,Helvetica,sans-serif;">found that M2 (pro-tumor) macrophages were found to highly overexpress the stress protein legumain, and the M1 (anti-tumor) did not. This meant that the tumor associated macrophages that promote angiogenesis as well as produce mitogenic factors for metastasis can be specifically targeted and destroyed due to their over-expression of legumain. The gene encoding legumain was fused to the c terminus of mutant polyubiquitin along with Kozak ATG (start codon) and pCMV. Now when this drug was administered the amount of M2 TAMs expressing legumain were completely abrogated from the tumor microenvironment. This removal of TAMs resulted in major downregulation of growth factors and pro-angiogenic factors. The destruction of the TAMs was explained to be as a result of calling cytotoxic T cells to the tumor to destroy them.

In the figure above, it is a representation of tumors with and without Tumor Associated Macrophages, the left is a tumor with Tumor Associated Macrophages as a part of the tumor microenvironment and it is clear that there is substantial amount of angiogenesis and inflammation. The left picture has been treated with the pLegumain drug and thus there is almost no, if little inflammation at the site.

"Re-educating" TAMS
In an articlepublished in 2008, Thorsten Hagemann and his fellow researchers identified a method to change tumor-associated macrophages into cytotoxic macrophages, which would then be able to target tumor cells as do normal MI macrophages. Hagemann et al. explained again what we already know - that the NF-κB signaling pathway is plays an important role in inflammation and tumor growth and progression. As such, they decided to target this pathway to reduce the pro-tumor effects of TAMs. They write, "When NF-κB signaling is inhibited specifically in TAMs, they become cytotoxic to tumor cells and switch to a 'classically' activated phenotype" (20).

In the experiment highlighted in the figure above, Hagemann et al. wanted to see if using IKKβ inhibition could re-educate macrophages and thus affect tumor progression. 5 weeks into the experiment, the mice started to develop tumors with deposits throughout the peritoneum. When researchers transfered IKKβ-targeted TAMs into these tumor-bearing mice, there was a significant decrease in tumor effects compared to mice with control macrophages. Measuring the IL-10, IL-12, and TNF-α levels 14 days after macrophage transfer, they tried to identify the TAM phenotype. The mice with IKKβ-targeted macrophages showed a significant change in cytokine production with a switch to an IL-10low IL-12high and TNF-αlow profile (the usual macrophage characteristics). Thus, it was concluded that through the targeting and inhibition of IKKβ in TAMs (which had been isolated from tumors), these tumor-associated macrophages convert their phenotype from pro-tumor M2 to the anti-tumor M1 type or macrophages.

=8. Conclusions=

Immunotherapy
Having looked at the two articles mentioned above, we believed that the second paper had a better method for combating tumor-associated macrophages and their effects. While the first article focused on destruction of TAMs, we felt that this alone was insufficient for curing cancer. Although petients experienced better prognosis and tumor growth halted, there is no guarantee that more macrophages will not turn into the pro-tumor type. In order to fully attack cancer cells, what needs to be done is switching tumor-associated macrophages from being pro-tumor to being anti-tumor. Thus, we concluded that the second article provided a better immunotherapy method.

However we did not feel then that the first method of destroying TAMs was not completely useless. Looking at the strategies that were used for removing TAMs, this would still be a helpful treatment that can be used in conjunction with normal cancer therapies such as radiation and chemotherapy to reduce both angiogenesis and tumor progression. Because although the cancer is not ablated from the body, the TAMs are. In cancer prognosis our inference is that the reduction in inflammation can improve patient prognosis, even if by a short period of time, because the invasive of cancer is significantly decreased. This was exhibited in the mice being treated by pLegumain. Thus, although we identified that this is not the best treatment for cancer patients because there other more effective methods of targeting the inflammation pathway; I think that this can be a last resort therapy to give patients more time and more cancer control.This experiment might also have some future implications with adaptive therapy. One issue we found with the experiment is that there needs to be a better understanding about the effects of pLegumain on the rest of the body, there is the capability of this drug to destroy cells with high Legumain expression, so we need to better understand where else Legumain is expressed and how to

In terms of the strategies used for the macrophage "reeducation" this method was greatly favored because it integrated the ideas expressed in the main articles we used as a reference (5). As mentioned before in the different traits of macrophages the changing of phenotype to manipulate cancer promoting properties is key. Thus, using this changing phenotype to our advantage is the best way to target the macrophage, because doing so would not only improve prognosis but defeat and destroy cancer cells as well. What made this immunotherapy procedure even more outstanding in our opinion was the targeting of the major pathway for cancer inflammation, the NF-κB. Targeting this pathway we felt would almost ensure that patient prognosis would improve.

All in all however, whether "reducating" or destroying, tumor associated macrophages play a critical role in the invasiveness of cancers. Thus targeting them for cancer treatment is important and may be more effective than traditional therapies because as Condeelis and Pollard suggest that macrophages make excellent targets for small molecule drugs as they "do not harbor malignant mutations and therefore have a stable genome" and are not likely to develop drug resistance.

Macrophage Tumor Promotion
The two main points that we drew from our research are as follows: 1. Inflammation is a key factor in tumor progression and metastasis. 2. Macrophages have an evolving phenotype that can be manipulated for immunotherapy.

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