Therapeutic Focus

Motivated by the potential to improve outcomes for patients with a wide range of cancer and inflammatory disorders, XTuit is leading a new approach by developing therapies that treat the disease microenvironment. This paradigm changing tactic focuses on normalizing the hijacked cellular and molecular machinery surrounding the disease site, returning it to its healthy physiologic function and preventing its use as a disease support system. By taking this holistic approach to therapeutic design, XTuit’s treatments work both alone and in tandem with standard of care agents to eliminate disease through the restoration of healthy tissue function in the microenvironment.

The
Microenvironment

The microenvironment, or stroma, is significantly altered in cancer and fibrotic disorders, contributing to and promoting the advancement of disease. The microenvironment is in a pro-inflammatory state and becomes stiff through deposition of extracellular matrix components, including collagen and hyaluronan, which are stabilized by matrix molecules. Moreover, stromal cells, including cancer-associated fibroblasts, stellate cells, and monocyte/macrophage-derived cells, expand and are reprogrammed to an active state. This activation results in up-regulation of several key signaling pathways in the stroma. As a consequence of these biological changes, there is increased solid stress in the stroma, resulting in vessel compression and ultimately hypoxia, as well as stroma-induced signaling to the tumor and inflammatory cells that can result in disease progression and treatment resistance to standard-of-care agents.

One critical need in accelerating cures for cancer is to solve the riddle of why some tumors resist therapy. Tumor cells, like cells from normal organs, are supported and nourished by connective tissue, also called stroma. The stromal microenvironment is comprised of both extracellular matrix (ECM) components, like collagen and hyaluronan and a number of stromal cell types, including fibroblasts, monocytes/macrophages, neutrophils, infiltrating immune cells, and natural killer (NK) cells. During cancer progression, various stromal cells become activated and proliferate in response to tumor cell signals, resulting in enhanced ECM deposition. The increased number of stromal cells and ECM lead to increased physical stress, causing vessel compression and restricted blood flow, making parts of the tumor hypoxic.  This “hypoxic core” harbors cancer stem cells and the hypoxic stress fosters genetic instability and mutations in the tumor cells. Tumor cell mutations and growth-promoting signaling from the activated stromal cells to the tumor cells eventually result in treatment resistance, further enhanced by the insufficient distribution of cancer therapy to the hypoxic regions due to vessel compression. Thus, non-malignant cells and associated factors are culprits in tumor growth, immune evasion and resistance to chemotherapy, radiation or immunotherapy.

Interestingly, the ECM component is more pronounced in ‘difficult-to-treat’ cancer types, like pancreatic, bladder, ovarian, breast and prostate cancers all of which have a relatively high mutation frequency.

Stromal changes during cancer development contribute to preventing efficient drug uptake and driving to therapeutic resistance. With a deep understanding of the biology of the stromal overproduction, we can design novel therapies able to reprogram or ‘normalize’ the altered cancer stroma, rendering it permissive to cancer therapy and/or to reinstate immune-surveillance necessary to control tumor growth.

The figure illustrates a sample of human pancreatic cancer with a relatively large number of tumor cells (green color) and relatively little extra-cellular matrix (red color) which may make this tumor somewhat less resistant to therapy.

As the tumor grows, stromal cells become activated and expand, and the extracellular matrix they produce typically become a more prominent part of the tumor. Capillary blood flow now becomes obstructed, reducing the access for cancer therapies to reach the tumor cells. Moreover, the activated stromal cells secrete growth promoting factors stimulating tumor cell growth.

The continued expansion of activated stromal cells and extracellular matrix results in a thickened, hardened stroma, called desmoplasia, which eventually causes hypoxia with not only limited cancer therapy access to the tumor cells, but also causes a non-permissive environment for infiltrating immune cells involved in immune surveillance.

In the most advanced stages, tumors can become quite fibrotic. Tumor cells now typically constitute a much smaller proportion relative to the stromal component. At this point, tumors typically have a hypoxic core with activated pro-inflammatory and tumor growth-promoting signals emanating from activated stromal cells, very few immune-competent cells, and limited access for cancer therapeutics, thus rendering the tumor almost impossible to treat successfully.

 

Fibrosis is a class of conditions caused by chronic inflammation arising from a failed repair response to injury or disease in numerous tissue types. Fibrosis represents a state of chronic dysregulation of extracellular matrix production and turnover, resulting in excessive accumulation of collagen, hyaluronan, and other fibrous connective tissue components in the diseased microenvironment that destroys normal organ architecture and function. Due to significant variability in the disease severity, fibrotic disorders can result in heterogeneous clinical outcomes that range from mild to severe and from chronic to acute. Ultimately, chronic inflammation with fibrosis can lead to organ failure and is also involved in cancer progression, both of which can be terminal.

 

Human liver tissue was stained with antibodies against markers of liver architecture. Cytokeratin 18 for hepatocytes (epithelial cells), a-Smooth Muscle Actin (SMA) for activated stellate cells and endothelial cells, and Iba1 for Kupffer cells (macrophages).

 

The pictures illustrate progression of fibrosis in human liver.

Normal (healthy) human liver

Hepatocytes make up a majority of the liver, interspersed with Kupffer cells and only very few smooth muscle actin (SMA)-positive stellate cells around blood vessels.

Early fibrosis/ NAFLD/ NASH

Non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) are common liver disorders characterized by lipid-filled, enlarged (“ballooning”) hepatocytes, SMA-positive activated stellate cells, and Kupffer cells in fibrotic areas

Advanced stage of fibrosis

The overall liver architecture is noticeably disrupted with multiple layers of SMA-positive activated stellate cells accumulating. Kupffer cells continue to accumulate around and within the increasingly fibrotic areas of the liver.

Cirrhosis

The liver structure is highly disrupted with islands of hepatocytes separated by significant fibrotic areas with SMA-positive activated stellate cells. Kupffer cells accumulate around and within the fibrotic areas.

Take a Peek Inside
The Microenvironment

Our proprietary biomarker platform allows us to quantitatively image multiple proteins, cell types, and tissue patterns simultaneously (so-called “multiplex biomarker imaging”). These biomarkers can help inform about the role, activity, and spatial relationships between various cell types in healthy and diseased tissues, quantify changes in protein expression and protein activity states, and ultimately serve to identify the patients that are likely to benefit from our treatments and to demonstrate mechanistic efficacy of our novel therapeutics in patients.

  • Pancreatic Cancer

    Pancreatic adenocarcinoma, which accounts for around 90% of pancreatic cancer, begins with proliferation of transformed cells arising from the pancreatic duct epithelial cells. At early stages there are predominantly tumor cells (Green Tab) with relatively fewer stromal cells (Red Tab) interspersed with newly formed capillaries and endothelial cell-derived angiogenic structures (Yellow Tab).

    Tumor Cells

    Tumor epithelial cells are the primary cancer cells in pancreatic adenocarcinoma. These cells multiply constantly, and have invasive properties. Tumor cells are detected using a fluorescently-labeled antibody against a cytokeratin, CK18,  which is a pan-epithelial marker.

    Stroma

    The stroma is made up of various non-epithelial cell types and extracellular matrix components produced by these cells. During tumor progression, fibroblasts and other cells become activated to produce more matrix and secrete tumor growth-promoting factors. These activated cells can be detected using a fluorescently-labeled antibody against alpha-smooth muscle actin (a-SMA).

    Endothelial

    Endothelial cells make up the inner lining of blood vessels and newly-formed capillaries. These cells can be detected using a fluorescently-labeled antibody against an endothelial cell surface marker called CD31.

    Nuclei

    All cells package their genetic information into nuclei. The nuclear shape and position in cells provides information on cell type, cell-cycle stage, and can reveal cellular processes such as the initiation of cell death or over-proliferation. Moreover, the nuclei allow for detailed imaging analysis through so-called subcellular localization quantitation, to measure the activity of various cellular proteins based on their location in either the nucleus or the cytoplasm.

  • Activated Stroma

    As the tumor progresses, stromal cells (Red Tab) become activated and proliferate, and the extracellular matrix they produce becomes a more prominent part of the tumor, ultimately squeezing the capillary structures (Yellow Tab). Consequently, capillary blood flow becomes increasingly obstructed, making the tumor more hypoxic and reducing delivery of cancer therapies. Moreover, the activated stromal cells secrete growth-promoting factors stimulating tumor cell growth.  The expansion of activated stromal cells and extracellular matrix result in a hardened stroma, a process called desmoplasia.

    Tumor Cells

    Tumor epithelial cells are the primary cancer cells in pancreatic adenocarcinoma. These cells multiply constantly, and have invasive properties. Tumor cells are detected using a fluorescently-labeled antibody against a cytokeratin, CK18,  which is a pan-epithelial marker.

    Stroma

    The stroma is made up of various non-epithelial cell types and extracellular matrix components produced by these cells. During tumor progression, fibroblasts and other cells become activated to produce more matrix and secrete tumor growth-promoting factors. These activated cells can be detected using a fluorescently-labeled antibody against alpha-smooth muscle actin (a-SMA).

    Endothelial

    Endothelial cells make up the inner lining of blood vessels and newly-formed capillaries. These cells can be detected using a fluorescently-labeled antibody against an endothelial cell surface marker called CD31.

    Nuclei

    All cells package their genetic information into nuclei. The nuclear shape and position in cells provides information on cell type, cell-cycle stage, and can reveal cellular processes such as the initiation of cell death or over-proliferation. Moreover, the nuclei allow for detailed imaging analysis through so-called subcellular localization quantitation, to measure the activity of various cellular proteins based on their location in either the nucleus or the cytoplasm.

  • Severe Desmoplasia

    In highly fibrotic areas tumor cells (Green Tab) constitute a much smaller proportion relative to the stromal component (Red Tab). Tumors now have a highly hypoxic core with activated pro-inflammatory and tumor growth-promoting signals emanating from activated stromal cells, very few immune-competent cells, and limited access for cancer therapeutics due to decreased perfusion from blood vessels (Yellow Tab). Consequently, the tumor is now almost impossible to treat successfully.

    Tumor Cells

    Tumor epithelial cells are the primary cancer cells in pancreatic adenocarcinoma. These cells multiply constantly, and have invasive properties. Tumor cells are detected using a fluorescently-labeled antibody against a cytokeratin, CK18,  which is a pan-epithelial marker.

    Fibroblast

    The stroma is made up of various non-epithelial cell types and extracellular matrix components produced by these cells. During tumor progression, fibroblasts and other cells become activated to produce more matrix and secrete tumor growth-promoting factors. These activated cells can be detected using a fluorescently-labeled antibody against alpha-smooth muscle actin (a-SMA).

    Endothelial

    Endothelial cells make up the inner lining of blood vessels and newly-formed capillaries. These cells can be detected using a fluorescently-labeled antibody against an endothelial cell surface marker called CD31.

    Nuclei

    All cells package their genetic information into nuclei. The nuclear shape and position in cells provides information on cell type, cell-cycle stage, and can reveal cellular processes such as the initiation of cell death or over-proliferation. Moreover, the nuclei allow for detailed imaging analysis through so-called subcellular localization quantitation, to measure the activity of various cellular proteins based on their location in either the nucleus or the cytoplasm.

  • Liver

    The liver comprises many cell types, primarily hepatocytes, with fewer numbers of sinusoidal endothelial cells, Kupffer cells, and quiescent hepatic stellate cells. In fibrotic disorders, the balance and activity of these various cell types is disrupted, with a decreased number of healthy hepatocytes, concomitant with increased pro-inflammatory signaling, activation of stellate cells, and an increased number of inflammatory Kupffer cells.

    Through the use of our multiplex biomarker imaging platform, we can objectively assess the quantitative changes in proteins, cell types, and tissue patterns and morphology during fibrotic disease progression.

    In this liver section, fibroblasts appear in red, hepatocytes appear in green, macrophages appear in yellow, and cell nuclei for all cell types appear in blue.

    Use the filter to see each cell type isolated.

    Hepatocytes

    Hepatocytes are the primary functional cells in the liver and responsible for the synthesis of lipoproteins, complement, serum albumin, fibrinogen, and prothrombin. They are critically important in the detoxification process for the body, removing some of the waste products of metabolism, as well as clearing drugs and environmental toxins.

    In this plate hepatocytes are stained with cytokeratin 18

    Fibroblasts

    Fibroblasts manufacture extracellular matrix (ECM) components and stabilizers such as collagen, hyaluronan, and connective tissue growth factor (CTGF). Hence, they are a critical element in establishing the normal microenvironment and providing structural support for other cell types. Fibroblasts are also critical mediators of the inflammatory response to tissue injury or infection. With chronic ongoing injury, as is seen for instance in  liver disease, the repeated cycles of inflammation and aberrant ECM deposition drive the development of fibrosis at the cost of healthy hepatocytes, which ultimately destroys organ function.

    In this plate the antibody against alpha-smooth muscle actin (SMA) reveals myofibroblasts surrounding the blood vessels.

    Macrophages

    During inflammation macrophages perform multiple roles. They serve as a first line of defense against pathogens and assist in stimulating the adaptive immune response by presenting antigens to T-cells. They can also decrease inflammation and begin tissue repair through secretion of anti-inflammatory cytokines. Macrophages reside in virtually every tissue type and when present in the liver are called Kupffer cells. In normal livers, Kupffer cells are regularly and evenly spaced, while in diseased livers, these cells proliferate in areas of injury and contribute to the advancement of fibrosis.

    In this plate macrophages are stained with Iba1

    Nuclei

    All cells package their genetic information into nuclei. The nuclear shape and position in cells provides information on cell type, cell-cycle stage, and can reveal cellular processes such as the initiation of cell death or over-proliferation. Moreover, the nuclei allow for detailed imaging analysis through so-called subcellular localization quantitation, to measure the activity of various cellular proteins.

    In this plate the cellular nuclei are stained with a DNA stain, DAPI

  • Fibrosis

    There are ballooning hepatocytes (Green Tab), but overall structure is fairly regular. There is a layer of SMA positive activated stellate cells (Red Tab) that secrete various extracellular matrix components. Kupffer cells (Yellow Tab) accumulate around and within fibrotic area. The biomarker platform can be used to assess the severity of the disease in a liver biopsy sample.

    Use the filter to see each cell type isolated.

    Hepatocytes

    Hepatocytes are the primary functional cells in the liver and responsible for the synthesis of lipoproteins, complement, serum albumin, fibrinogen, and prothrombin. They are critically important in the detoxification process for the body, removing some of the waste products of metabolism, as well as clearing drugs and environmental toxins.

    In this plate hepatocytes are stained with cytokeratin 18

    Fibroblasts

    Fibroblasts manufacture extracellular matrix (ECM) components and stabilizers such as collagen, hyaluronan, and connective tissue growth factor (CTGF). Hence, they are a critical element in establishing the normal microenvironment and providing structural support for other cell types. Fibroblasts are also critical mediators of the inflammatory response to tissue injury or infection. With chronic ongoing injury, as is seen for instance in  liver disease, the repeated cycles of inflammation and aberrant ECM deposition drive the development of fibrosis at the cost of healthy hepatocytes, which ultimately destroys organ function.

    In this plate the antibody against alpha-smooth muscle actin (SMA) reveals myofibroblasts surrounding the blood vessels.

    Macrophages

    During inflammation macrophages perform multiple roles. They serve as a first line of defense against pathogens and assist in stimulating the adaptive immune response by presenting antigens to T-cells. They can also decrease inflammation and begin tissue repair through secretion of anti-inflammatory cytokines. Macrophages reside in virtually every tissue type and when present in the liver are called Kupffer cells. In normal livers, Kupffer cells are regularly and evenly spaced, while in diseased livers, these cells proliferate in areas of injury and contribute to the advancement of fibrosis.

    In this plate macrophages are stained with Iba1

    Nuclei

    All cells package their genetic information into nuclei. The nuclear shape and position in cells provides information on cell type, cell-cycle stage, and can reveal cellular processes such as the initiation of cell death or over-proliferation. Moreover, the nuclei allow for detailed imaging analysis through so-called subcellular localization quantitation, to measure the activity of various cellular proteins.

    In this plate the cellular nuclei are stained with a DNA stain, DAPI

  • Cirrhosis

    Hepatocytes structure is highly disrupted with separate islands of cells (Green Tab). Tissue is filled with massive amount of fibrotic areas with SMA positive activated stellate cells. Kupffer cells (Yellow Tab) accumulate around and within fibrotic area (Red Tab). Our biomarker platform allows accurate resolution and quantitation of these diverse cell types, and can be used to monitor treatment efficacy in discovery or clinical research.

    Use the filter to see each cell type isolated.

    Hepatocytes

    Hepatocytes are the primary functional cells in the liver and responsible for the synthesis of lipoproteins, complement, serum albumin, fibrinogen, and prothrombin. They are critically important in the detoxification process for the body, removing some of the waste products of metabolism, as well as clearing drugs and environmental toxins.

    In this plate hepatocytes are stained with cytokeratin 18

    Fibroblasts

    Fibroblasts manufacture extracellular matrix (ECM) components and stabilizers such as collagen, hyaluronan, and connective tissue growth factor (CTGF). Hence, they are a critical element in establishing the normal microenvironment and providing structural support for other cell types. Fibroblasts are also critical mediators of the inflammatory response to tissue injury or infection. With chronic ongoing injury, as is seen for instance in  liver disease, the repeated cycles of inflammation and aberrant ECM deposition drive the development of fibrosis at the cost of healthy hepatocytes, which ultimately destroys organ function.

    In this plate the antibody against alpha-smooth muscle actin (SMA) reveals myofibroblasts surrounding the blood vessels.

    Macrophages

    During inflammation macrophages perform multiple roles. They serve as a first line of defense against pathogens and assist in stimulating the adaptive immune response by presenting antigens to T-cells. They can also decrease inflammation and begin tissue repair through secretion of anti-inflammatory cytokines. Macrophages reside in virtually every tissue type and when present in the liver are called Kupffer cells. In normal livers, Kupffer cells are regularly and evenly spaced, while in diseased livers, these cells proliferate in areas of injury and contribute to the advancement of fibrosis.

    In this plate macrophages are stained with Iba1

    Nuclei

    All cells package their genetic information into nuclei. The nuclear shape and position in cells provides information on cell type, cell-cycle stage, and can reveal cellular processes such as the initiation of cell death or over-proliferation. Moreover, the nuclei allow for detailed imaging analysis through so-called subcellular localization quantitation, to measure the activity of various cellular proteins.

    In this plate the cellular nuclei are stained with a DNA stain, DAPI