Chimeric Antigen Cytotoxic Receptors for In Vivo Engineering of Tumor-Targeting NK Cells.

Chimeric Ag receptor (CAR) NK cells are challenging to manufacture and fail to achieve consistent tumor infiltration and sustained cytolytic function in the tumor microenvironment. In vivo engineering of NK cells using mRNA-based CAR delivery may overcome these issues. In this study, we developed an in vivo programming method by designing CARs that leverage the biology of NK cell receptors for cell type-specific expression and function. These CARs were engineered by fusion of a tumor recognition domain with the natural cytotoxic receptor family including NKp30, NKp44, and NKp46. Our results demonstrated that these natural cytotoxic receptor-based CARs can engage endogenous signaling adaptors to effectively activate human NK cells for tumor lysis and cytokine production. Specifically, we discovered that stable expression of an NKp44-based CAR was contingent on the presence of the immune cell-specific signaling adaptor DAP12. This innovative strategy facilitates direct in situ programming of NK cells, enhancing safety and minimizing off-target effects in nontargeted, healthy tissues.

Basement membrane damage by ROS- and JNK-mediated Mmp2 activation drives macrophage recruitment to overgrown tissue.

Macrophages are a major immune cell type infiltrating tumors and promoting tumor growth and metastasis. To elucidate the mechanism of macrophage recruitment, we utilize an overgrowth tumor model ("undead" model) in larval Drosophila imaginal discs that are attached by numerous macrophages. Here we report that changes to the microenvironment of the overgrown tissue are important for recruiting macrophages. First, we describe a correlation between generation of reactive oxygen species (ROS) and damage of the basement membrane (BM) in all neoplastic, but not hyperplastic, models examined. ROS and the stress kinase JNK mediate the accumulation of matrix metalloproteinase 2 (Mmp2), damaging the BM, which recruits macrophages to the tissue. We propose a model where macrophage recruitment to and activation at overgrowing tissue is a multi-step process requiring ROS- and JNK-mediated Mmp2 upregulation and BM damage. These findings have implications for understanding the role of the tumor microenvironment for macrophage activation.

Two Sides of the Same Coin - Compensatory Proliferation in Regeneration and Cancer.

Apoptosis has long been regarded as a tumor suppressor mechanism and evasion from apoptosis is considered to be one hallmark of cancer. However, this principle is not always consistent with clinical data which often illustrate a correlation between apoptosis and poor prognosis. Work in the last 15 years has provided an explanation for this apparent paradox. Apoptotic cells communicate with their environment and can produce signals which promote compensatory proliferation of surviving cells. This behavior of apoptotic cells is important for tissue regeneration in several model organisms, ranging from hydra to mammals. However, it may also play an important feature for tumorigenesis and tumor relapse. Several distinct forms of apoptosis-induced compensatory proliferation (AiP) have been identified, many of which involve reactive oxygen species (ROS) and immune cells. One type of AiP, "undead" AiP, in which apoptotic cells are kept in an immortalized state and continuously divide, may have particular relevance for tumorigenesis. Furthermore, given that chemo- and radiotherapy often aim to kill tumor cells, an improved understanding of the effects of apoptotic cells on the tumor and the tumor environment is of critical importance for the well-being of the patient. In this review, we summarize the current knowledge of AiP and focus our attention on recent findings obtained in Drosophila and other model organisms, and relate them to tumorigenesis.

An unexpected friend - ROS in apoptosis-induced compensatory proliferation: Implications for regeneration and cancer.

Apoptosis-induced compensatory proliferation (AiP) is a form of compensatory proliferation that is triggered by apoptotic cell death to maintain tissue homeostasis. As such, AiP is essential for many tissue repair processes including regeneration. The apoptotic effectors, termed caspases, not only execute apoptosis, but are also directly involved in the generation of the signals required for AiP. Reactive oxygen species (ROS) play an important role for regenerative processes. Recently, it was shown in Drosophila that apoptotic caspases can mediate the generation of ROS for promoting AiP. This review summarizes and discusses these findings in the context of regenerative processes and cancer.

The beneficial role of extracellular reactive oxygen species in apoptosis-induced compensatory proliferation.

Apoptosis-induced proliferation (AiP) maintains tissue homeostasis following massive stress-induced cell death. During this phenomenon, dying cells induce proliferation of the surviving cells to compensate for the tissue loss, and thus restore organ size. Along with wound healing and tissue regeneration, AiP also contributes to tumor repopulation following radiation or chemotherapy. There are several models of AiP. Using an "undead" AiP model that causes hyperplastic overgrowth of Drosophila epithelial tissue, we recently demonstrated that extracellular reactive oxygen species (eROS) are produced by undead epithelial cells, and are necessary for inducing AiP and overgrowth. Furthermore, hemocytes, the Drosophila blood cells, are seen adjacent to the undead epithelial tissue, and may secrete the TNF ortholog Eiger that signals through the TNF receptor to active Jun-N-terminal kinase (JNK) in the undead tissue and induce proliferation. We propose that undead epithelial tissue triggers an inflammatory response that resembles recruitment of macrophages to human epithelial tumors, and that these tumor-associated macrophages release signals for proliferation and tumor growth of the epithelium. This Extra View article summarizes these recent findings with a focus on the role of eROS for promoting regeneration and inflammation-induced tumorigenesis.

Ral-Arf6 crosstalk regulates Ral dependent exocyst trafficking and anchorage independent growth signalling.

Integrin dependent regulation of growth factor signalling confers anchorage dependence that is deregulated in cancers. Downstream of integrins and oncogenic Ras the small GTPase Ral is a vital mediator of adhesion dependent trafficking and signalling. This study identifies a novel regulatory crosstalk between Ral and Arf6 that controls Ral function in cells. In re-adherent mouse fibroblasts (MEFs) integrin dependent activation of RalA drives Arf6 activation. Independent of adhesion constitutively active RalA and RalB could both however activate Arf6. This is further conserved in oncogenic H-Ras containing bladder cancer T24 cells, which express anchorage independent active Ral that supports Arf6 activation. Arf6 mediates active Ral-exocyst dependent delivery of raft microdomains to the plasma membrane that supports anchorage independent growth signalling. Accordingly in T24 cells the RalB-Arf6 crosstalk is seen to preferentially regulate anchorage independent Erk signalling. Active Ral we further find uses a Ral-RalBP1-ARNO-Arf6 pathway to mediate Arf6 activation. This study hence identifies Arf6, through this regulatory crosstalk, to be a key downstream mediator of Ral isoform function along adhesion dependent pathways in normal and cancer cells.

Extracellular Reactive Oxygen Species Drive Apoptosis-Induced Proliferation via Drosophila Macrophages.

Apoptosis-induced proliferation (AiP) is a compensatory mechanism to maintain tissue size and morphology following unexpected cell loss during normal development, and may also be a contributing factor to cancer and drug resistance. In apoptotic cells, caspase-initiated signaling cascades lead to the downstream production of mitogenic factors and the proliferation of neighboring surviving cells. In epithelial cells of Drosophila imaginal discs, the Caspase-9 ortholog Dronc drives AiP via activation of Jun N-terminal kinase (JNK); however, the specific mechanisms of JNK activation remain unknown. Here we show that caspase-induced activation of JNK during AiP depends on an inflammatory response. This is mediated by extracellular reactive oxygen species (ROSs) generated by the NADPH oxidase Duox in epithelial disc cells. Extracellular ROSs activate Drosophila macrophages (hemocytes), which in turn trigger JNK activity in epithelial cells by signaling through the tumor necrosis factor (TNF) ortholog Eiger. We propose that in an immortalized ("undead") model of AiP, signaling back and forth between epithelial disc cells and hemocytes by extracellular ROSs and TNF/Eiger drives overgrowth of the disc epithelium. These data illustrate a bidirectional cell-cell communication pathway with implication for tissue repair, regeneration, and cancer.