Final Report Summary - CAFTECH (Unraveling the therapeutic potential of cancer-associated fibroblasts (CAF) in cancer using cell-specific targeting technology)
Publishable summary
Cancer is yet one of the most devastating diseases in the clinic accounting 7.5 million deaths per year world-wide. Tumor growth has traditionally been considered to be mostly dependent on properties of the malignant cells. However, it is now clear that additional molecular and cellular components of the tumor microenvironment also dictate tumor behaviors. Tumor stroma is the main component for these activities. Within stroma, cancer-associated fibroblasts (CAFs) are the most important cell type responsible for inducing tumorigenesis, angiogenesis and metastasis. PDGFbeta receptor is commonly, but variably, expressed on CAF. We have developed a novel CAF-targeting technology using a PDGFbetaR-binding carrier (HPC). Since PDGFbetaR is much strongly expressed on these cells than normal fibroblasts or other cell types, this carrier becomes rather selective for CAFs. HPC is made up of PDGFbetaR−binding cyclic peptide conjugated to human serum albumin. We have delivered doxorubicin to tumors using this carrier.
The main objectives for this project were to investigate 1) the potential role of CAFs in tumor mechanisms using CAF-targeting approach and 2) the importance of various signaling pathways in CAF functions.
To investigate the role of CAFs in tumor mechanisms, we applied CAF targeting approach by targeting interferon gamma (IFNy), an antifibrotic cytokine, to CAFs in tumor to inactivate them. In this way, CAF’s function could be inhibited and the role of CAFs on different tumor mechanisms could be studied. We first synthesized a targeted construct of IFNy by conjugating it to HPC carrier using biochemical linkage. We evaluated the binding of the construct to the target receptor using immunofluorescence methods and the efficacy in fibroblasts using real-time PCR and immunocytochemistry. To study the role of CAFs in vivo on tumor growth and angiogenesis, we used B16 melanoma tumor model and treated animals with the targeted IFNy construct intravenously and studied the role of CAFs and other stromal cells in relevance of the effects.
To investigate the importance of signaling pathways in CAFs and to find new targets on the CAFs in order to develop CAF-specific therapies, we performed a gene microarray study on a large collection of normal fibroblasts and CAFs isolated from patient tumor specimens. These fibroblasts derived from tumors were cultured and total RNA was isolated from these cells. These samples were then processed for the gene microarray analyses. The microarray data is analyzed for finding the new pathways.
Results: The targeted IFNy construct (HPC-IFNy) was successfully synthesized as determined with physicochemical characterization and in vitro studies in fibroblasts. In B16-F10 subcutaneous tumor-bearing mice, treatment with the targeted interferon gamma construct significantly reduced the progression of this malignant tumor while the nontargeted IFNy and the carrier did not show any inhibitory effect. We further explored the effect of the targeted construct on stromal cells and found that alpha-SMA-positive cells (CAFs) were markedly less prevalent in the targeted IFNy-treated tumors compared to control tumors. This showed that inhibition of CAF activity reduced the tumor growth indicating their role in tumor growth and progression. We examined the tumors for angiogenesis using immunohistochemical staining for CD31 (a marker for endothelial cells) and found that there was a reduction in angiogenesis with the targeted construct. In addition, we assured that these effects were not displayed through immunomodulatory effects of IFNy. These data suggest that CAFs play an important role in the regulation of tumor growth and angiogenesis.
In the other subproject, the gene microarray analyses was performed on normal fibroblasts and CAFs from breast, prostrate, pancreatic and colorectal carcinoma. Different types of cluster analyses were performed and we found that cells from each tumor type formed their own cluster suggesting that tumor type is a dominant factor in defining CAF characteristics. We are currently analyzing the data to investigate differentially expressed targets on CAFs and novel pathways responsible for CAF characteristics.
Potential impact and use: These data clearly indicate that CAFs are key cell type regulating tumor processes such as angiogenesis and tumor growth. These data is highly important to understand the complexity of tumor stromal biology. In addition, the contribution of cell-specific drug targeting field to study biology will certainly make impact in this field. Since we found remarkable tumor inhibiting effects of our targeted construct, this construct can potentially be developed as a novel therapeutic agent for cancer. Furthermore, our gene microarray study will disclose new targets which will be further explored for their applications in tumor biology and therapeutics.
Cancer is yet one of the most devastating diseases in the clinic accounting 7.5 million deaths per year world-wide. Tumor growth has traditionally been considered to be mostly dependent on properties of the malignant cells. However, it is now clear that additional molecular and cellular components of the tumor microenvironment also dictate tumor behaviors. Tumor stroma is the main component for these activities. Within stroma, cancer-associated fibroblasts (CAFs) are the most important cell type responsible for inducing tumorigenesis, angiogenesis and metastasis. PDGFbeta receptor is commonly, but variably, expressed on CAF. We have developed a novel CAF-targeting technology using a PDGFbetaR-binding carrier (HPC). Since PDGFbetaR is much strongly expressed on these cells than normal fibroblasts or other cell types, this carrier becomes rather selective for CAFs. HPC is made up of PDGFbetaR−binding cyclic peptide conjugated to human serum albumin. We have delivered doxorubicin to tumors using this carrier.
The main objectives for this project were to investigate 1) the potential role of CAFs in tumor mechanisms using CAF-targeting approach and 2) the importance of various signaling pathways in CAF functions.
To investigate the role of CAFs in tumor mechanisms, we applied CAF targeting approach by targeting interferon gamma (IFNy), an antifibrotic cytokine, to CAFs in tumor to inactivate them. In this way, CAF’s function could be inhibited and the role of CAFs on different tumor mechanisms could be studied. We first synthesized a targeted construct of IFNy by conjugating it to HPC carrier using biochemical linkage. We evaluated the binding of the construct to the target receptor using immunofluorescence methods and the efficacy in fibroblasts using real-time PCR and immunocytochemistry. To study the role of CAFs in vivo on tumor growth and angiogenesis, we used B16 melanoma tumor model and treated animals with the targeted IFNy construct intravenously and studied the role of CAFs and other stromal cells in relevance of the effects.
To investigate the importance of signaling pathways in CAFs and to find new targets on the CAFs in order to develop CAF-specific therapies, we performed a gene microarray study on a large collection of normal fibroblasts and CAFs isolated from patient tumor specimens. These fibroblasts derived from tumors were cultured and total RNA was isolated from these cells. These samples were then processed for the gene microarray analyses. The microarray data is analyzed for finding the new pathways.
Results: The targeted IFNy construct (HPC-IFNy) was successfully synthesized as determined with physicochemical characterization and in vitro studies in fibroblasts. In B16-F10 subcutaneous tumor-bearing mice, treatment with the targeted interferon gamma construct significantly reduced the progression of this malignant tumor while the nontargeted IFNy and the carrier did not show any inhibitory effect. We further explored the effect of the targeted construct on stromal cells and found that alpha-SMA-positive cells (CAFs) were markedly less prevalent in the targeted IFNy-treated tumors compared to control tumors. This showed that inhibition of CAF activity reduced the tumor growth indicating their role in tumor growth and progression. We examined the tumors for angiogenesis using immunohistochemical staining for CD31 (a marker for endothelial cells) and found that there was a reduction in angiogenesis with the targeted construct. In addition, we assured that these effects were not displayed through immunomodulatory effects of IFNy. These data suggest that CAFs play an important role in the regulation of tumor growth and angiogenesis.
In the other subproject, the gene microarray analyses was performed on normal fibroblasts and CAFs from breast, prostrate, pancreatic and colorectal carcinoma. Different types of cluster analyses were performed and we found that cells from each tumor type formed their own cluster suggesting that tumor type is a dominant factor in defining CAF characteristics. We are currently analyzing the data to investigate differentially expressed targets on CAFs and novel pathways responsible for CAF characteristics.
Potential impact and use: These data clearly indicate that CAFs are key cell type regulating tumor processes such as angiogenesis and tumor growth. These data is highly important to understand the complexity of tumor stromal biology. In addition, the contribution of cell-specific drug targeting field to study biology will certainly make impact in this field. Since we found remarkable tumor inhibiting effects of our targeted construct, this construct can potentially be developed as a novel therapeutic agent for cancer. Furthermore, our gene microarray study will disclose new targets which will be further explored for their applications in tumor biology and therapeutics.