Michael Timaner

Macrophage-Induced Lymphangiogenesis and Metastasis following Paclitaxel Chemotherapy Is Regulated by VEGFR3

Summary While chemotherapy strongly restricts or reverses tumor growth, the response of host tissue to therapy can counteract its anti-tumor activity by promoting tumor re-growth and/or metastases, thus limiting therapeutic efficacy. Here, we show that vascular endothelial growth factor receptor 3 (VEGFR3)-expressing macrophages infiltrating chemotherapy-treated tumors play a significant role in metastasis. They do so in part by inducing lymphangiogenesis as a result of cathepsin release, leading to VEGF-C upregulation by heparanase. We found that macrophages from chemotherapy-treated mice are sufficient to trigger lymphatic vessel activity and structure in naive tumors in a VEGFR3-dependent manner. Blocking VEGF-C/VEGFR3 axis inhibits the activity of chemotherapy-educated macrophages, leading to reduced lymphangiogenesis in treated tumors. Overall, our results suggest that disrupting the VEGF-C/VEGFR3 axis not only directly inhibits lymphangiogenesis but also blocks the pro-metastatic activity of macrophages in chemotherapy-treated mice.

Dequalinium blocks macrophage-induced metastasis following local radiation

A major therapeutic obstacle in clinical oncology is intrinsic or acquired resistance to therapy, leading to subsequent relapse. We have previously shown that systemic administration of different cytotoxic drugs can induce a host response that contributes to tumor angiogenesis, regrowth and metastasis. Here we characterize the host response to a single dose of local radiation, and its contribution to tumor progression and metastasis. We show that plasma from locally irradiated mice increases the migratory and invasive properties of colon carcinoma cells. Furthermore, locally irradiated mice intravenously injected with CT26 colon carcinoma cells succumb to pulmonary metastasis earlier than their respective controls. Consequently, orthotopically implanted SW480 human colon carcinoma cells in mice that underwent radiation, exhibited increased metastasis to the lungs and liver compared to their control tumors. The irradiated tumors exhibited an increase in the colonization of macrophages compared to their respective controls; and macrophage depletion in irradiated tumor-bearing mice reduces the number of metastatic lesions. Finally, the anti-tumor agent, dequalinium-14, in addition to its anti-tumor effect, reduces macrophage motility, inhibits macrophage infiltration of irradiated tumors and reduces the extent of metastasis in locally irradiated mice. Overall, this study demonstrates the adverse effects of local radiation on the host that result in macrophage-induced metastasis.

The antiangiogenic role of the pro-inflammatory cytokine interleukin-31

Pro-inflammatory cytokines in the tumor microenvironment are known for their ability to either inhibit or promote cancer progression. Here we evaluated the role of Interleukin-31 (IL31), a protein belonging to the pro-inflammatory IL-6 cytokine family which has been characterized in autoimmune disease, in tumorigenesis. We show that IL31 and its receptor, IL31RA, are highly expressed in various human and mouse cancer cell lines, as well as in tumor specimens from cancer patients. MC38 murine colon carcinoma cells depleted of IL31 exhibit an increase in invasive and migratory properties in vitro, effects that are reversed by supplementing the cells with exogenous IL31. In vivo, IL31-depleted MC38 tumor cells implanted to mice grow faster than control tumors. In contrast, MC38 tumor-bearing mice infused with recombinant IL31, exhibit a significant reduction in tumor growth than control mice. Furthermore, IL31 infusion reduces the number of metastatic lesions in the lungs of mice bearing 4T1 murine metastatic breast carcinoma. Lastly, injecting tumor-bearing, chemotherapy-treated mice with a long-lived IL31-IgG fusion protein reduces tumor growth, angiogenesis and pulmonary metastasis to a greater extent than when chemotherapy is used alone. The IL31 anti-tumor activity is explained, in part, by the anti-angiogenic effects demonstrated both in vitro and in vivo highlighting the potential use of IL31 as an anti-cancer drug.

Bortezomib-induced pro-inflammatory macrophages as a potential factor limiting anti-tumour efficacy

Multiple myeloma (MM) is a chronic progressive malignancy of plasma cells. Although treatment with the novel proteasome inhibitor, bortezomib, significantly improves patient survival, some patients fail to respond due to the development of de novo resistance. We have previously shown that cytotoxic drugs can induce pro‐tumorigenic host‐mediated effects which contribute to tumour re‐growth and metastasis, and thus limit anti‐tumour efficacy. However, such effects and their impact on tumour cell aggressiveness have not been investigated using cytostatic agents such as bortezomib. Here we show that plasma from bortezomib‐treated mice significantly increases migration, viability and proliferation of MM cells in vitro, compared to plasma from vehicle treated mice. In vivo, bortezomib induces the mobilization of pro‐angiogenic bone marrow cells. Furthermore, mice treated with bortezomib and subsequently were used as recipients for an injection of MM cells succumb to MM earlier than mice treated with the vehicle. We show that bortezomib promotes pro‐inflammatory macrophages which account for MM cell aggressiveness, an effect which is partially mediated by interleukin‐16. Accordingly, co‐inoculation of MM cells with pro‐inflammatory macrophages from bortezomib‐treated mice accelerates MM disease progression. Taken together, our results suggest that, in addition to the known effective anti‐tumour activity of bortezomib, host‐driven pro‐tumorigenic effects generated in response to treatment can promote MM aggressiveness, and thus may contribute to the overall limited efficacy. Copyright

Therapy-Educated Mesenchymal Stem Cells Enrich for Tumor-Initiating Cells

Stromal cells residing in the tumor microenvironment contribute to the development of therapy resistance. Here we show that chemotherapy-educated mesenchymal stem cells (MSC) promote therapy resistance via cross-talk with tumor-initiating cells (TIC), a resistant tumor cell subset that initiates tumorigenesis and metastasis. In response to gemcitabine chemotherapy, MSCs colonized pancreatic adenocarcinomas in large numbers and resided in close proximity to TICs. Furthermore, gemcitabine-educated MSCs promoted the enrichment of TICs in vitro and enhance tumor growth in vivo These effects were dependent on the secretion of CXCL10 by gemcitabine-educated MSCs and subsequent activation of the CXCL10-CXCR3 axis in TICs. In an orthotopic pancreatic tumor model, targeting TICs using nanovesicles (called nanoghosts) derived from MSC membranes and loaded with a CXCR3 antagonist enhanced therapy outcome and delayed tumor regrowth when administered in combination with gemcitabine. Overall, our results establish a mechanism through which MSCs promote chemoresistance, and propose a novel drug delivery system to target TICs and overcome this resistance.Significance: These results establish a mechanism by which mesenchyme stem cells in the tumor microenvironment promote chemoresistance, and they propose a novel drug delivery system to overcome this challenge. Cancer Res; 78(5); 1253-65. ©2018 AACR.

Analysis of the Stromal Cellular Components of the Solid Tumor Microenvironment Using Flow Cytometry

The tumor microenvironment consists of a variety of cell types. The contribution of each cell type to the tumor is an emerging subject in the field of cancer research. Here, we describe protocols for dissociating tumor tissues and Matrigel plugs into single cells for further analysis by flow cytometry. These protocols can be used for evaluating the cellular component of solid tumors from human or mouse origin or Matrigel plugs implanted in mice. The protocols describe the dissociation of tumor tissue with or without dissociation automatic devices. Subsequently, the use of flow cytometry for immunophenotypic analysis of host cells found in the tumor microenvironment, including myeloid derived suppressor cells, endothelial cells, and macrophages is provided. These methods can be used to broaden our understanding of the cross‐talk between tumor and host cells in the tumor microenvironment.

Host JDP2 expression in the bone marrow contributes to metastatic spread

The c-Jun Dimerization Protein 2, JDP2, is a basic leucine zipper protein member of the activator protein-1 (AP-1) family of transcription factors. JDP2 typically suppresses gene transcription through multiple mechanisms and plays a dual role in multiple cellular processes, including cell differentiation and proliferation which is dependent on AP-1 function. Whereas the role of JDP2 expression within cancer cells has been studied, its role in stromal cells at the tumor microenvironment is largely unknown. Here we show that mice lacking JDP2 (JDP2−/−) display a reduced rate of metastasis in Lewis lung carcinoma (LLC) and polyoma middle T-antigen (PyMT) breast carcinoma mouse models. The replacement of wild-type bone marrow derived cells (BMDCs) with JDP2-deficient BMDCs recapitulates the metastatic phenotype of JDP2−/− tumor-bearing mice. In vitro, conditioned medium of wild-type BMDCs significantly potentiates the migration and invasion capacity of LLC cells as compared to that of JDP2−/− BMDCs. Furthermore, wild-type BMDCs secrete CCL5, a chemokine known to contribute to metastasis, to a greater extent than JDP2−/− BMDCs. The supplementation of CCL5 in JDP2−/− BMDC conditioned medium was sufficient to potentiate the invasion capacity of LLC. Overall, this study suggests that JDP2-expressing BMDCs within the tumor microenvironment contribute to metastatic spread.

Dose- and time-dependence of the host-mediated response to paclitaxel therapy: a mathematical modeling approach

It has recently been suggested that pro-tumorigenic host-mediated processes induced in response to chemotherapy counteract the anti-tumor activity of therapy, and thereby decrease net therapeutic outcome. Here we use experimental data to formulate a mathematical model describing the host response to different doses of paclitaxel (PTX) chemotherapy as well as the duration of the response. Three previously described host-mediated effects are used as readouts for the host response to therapy. These include the levels of circulating endothelial progenitor cells in peripheral blood and the effect of plasma derived from PTX-treated mice on migratory and invasive properties of tumor cells in vitro. A first set of mathematical models, based on basic principles of pharmacokinetics/pharmacodynamics, did not appropriately describe the dose-dependence and duration of the host response regarding the effects on invasion. We therefore provide an alternative mathematical model with a dose-dependent threshold, instead of a concentration-dependent one, that describes better the data. This model is integrated into a global model defining all three host-mediated effects. It not only precisely describes the data, but also correctly predicts host-mediated effects at different doses as well as the duration of the host response. This mathematical model may serve as a tool to predict the host response to chemotherapy in cancer patients, and therefore may be used to design chemotherapy regimens with improved therapeutic outcome by minimizing host mediated effects.

A new screening method for ATP-independent kinase inhibitors identifies repurposed anti-cancer drugs

In the era of targeted drugs for cancer, small molecule kinase inhibitors have been designed to target specific downstream molecules in tumor-promoting signaling pathways; however, such compounds often exhibit high toxicity profiles due in part to their off-target effects [1]. This limits the drug dosage that can be used safely, and concomitantly, decreases drug efficacy. Therefore, there is a clear need to identify inhibitory molecules with high affinity and greater specificity against their target. In this issue of EBioMedicine, Cheng et al., describe a novel and highly efficient drug screening technology for the identification of potent and specific molecules that inhibit Dbf4-dependent kinase (DDK) [10]. DDK as well as cyclin dependent kinases (CDKs) are proteins associated with cell cycle. As such, they have been considered as major targets for drug screening [2]. Among these proteins, cell division cycle 7-related protein kinase (Cdc7) is a serine threonine kinase with an important role in initiating DNA replication and cell cycle control in eukaryotic cells. Cdc7 kinase activity is regulated by Dbf4 activation unit and in turn promotes the initiation of DNA synthesis by phosphorylation of MCM2–7 helicase complex throughout the S phase [3]. Indeed, in line with its crucial function, Cdc7 was shown to be overexpressed in multiple malignant conditions, including breast, colon, lung, ovarian carcinomas aswell as diffuse large B cell lymphoma, and oral squamous cell carcinoma (OSCC) [4]. Besides DNA replication initiation, Cdc7/Dbf4 kinase maintains DNA damage tolerance and increases cancer cell survival [3]. Noteworthy, a strong correlation between the Cdc7/Dbf4 kinase expression levels and mutational burden of the tumor was recently demonstrated, suggesting that increased DDK levels promote mutagenesis and contribute to chemoresistance [5]. Therefore, Cdc7 inhibitors can potentially serve as a useful therapy for cancer. Indeed, in recent years, a large number of Cdc7 inhibitors have been developed to blunt pro-tumorigenic activity. For instance, XL413, a benzofuropyrimidinone selective ATP-competitive Cdc7 inhibitor was shown to reduce viability and proliferation of OSCC cells and enhance the cytotoxic effect of cisplatin and 5-fluorouracil [4]. However, like