ABSTRACT
Cytotoxic immune cells, including T lymphocytes (CTLs) and natural killer (NK) cells, are essential components of the host response against tumors. CTLs and NK cells secrete granzyme A (GzmA) upon recognition of cancer cells; however, there are very few tools that can detect physiological levels of active GzmA with high spatiotemporal resolution. Herein, we report the rational design of the near-infrared fluorogenic substrates for human GzmA and mouse GzmA. These activity-based probes display very high catalytic efficiency and selectivity over other granzymes, as shown in tissue lysates from wild-type and GzmA knock-out mice. Furthermore, we demonstrate that the probes can image how adaptive immune cells respond to antigen-driven recognition of cancer cells in real time.
Subject(s)
Fluorescent Dyes , T-Lymphocytes, Cytotoxic , Animals , Humans , Mice , Granzymes , Killer Cells, Natural , Mice, KnockoutABSTRACT
Granzymes (Gzms), a family of serine proteases, expressed by immune and nonimmune cells, present perforin-dependent and independent intracellular and extracellular functions. When released in the extracellular space, GzmA, with trypsin-like activity, is involved in the pathophysiology of different inflammatory diseases. However, there are no validated specific systems to detect active forms of extracellular GzmA, making it difficult to assess its biological relevance and potential use as a biomarker. Here, we have developed fluorescence-energy resonance-transfer (FRET)-based peptide probes (FAM-peptide-DABCYL) to specifically detect GzmA activity in tissue samples and biological fluids in both mouse and human samples during inflammatory diseases. An initial probe was developed and incubated with GzmA and different proteases like GzmB and others with similar cleavage specificity as GzmA like GzmK, thrombin, trypsin, kallikrein, or plasmin. After measuring fluorescence, the probe showed very good specificity and sensitivity for human and mouse GzmA when compared to GzmB, its closest homologue GzmK, and with thrombin. The specificity of this probe was further refined by incubating the samples in a coated plate with a GzmA-specific antibody before adding the probe. The results show a high specific detection of soluble GzmA even when compared with other soluble proteases with very similar cleavage specificity like thrombin, GzmK, trypsin, kallikrein, or plasmin, which shows nearly no fluorescence signal. The high specific detection of GzmA was validated, showing that using pure proteins and serum and tissue samples from GzmA-deficient mice presented a significant reduction in the signal compared with WT mice. The utility of this system in humans was confirmed, showing that GzmA activity was significantly higher in serum samples from septic patients in comparison with healthy donors. Our results present a new immunoprobe with utility to detect extracellular GzmA activity in different biological fluids, confirming the presence of active forms of the soluble protease in vivo during inflammatory and infectious diseases.
ABSTRACT
Background: Docetaxel remains the standard treatment for metastatic castration-resistant prostate cancer (mCRPC). However, resistance frequently emerges as a result of hyperactivation of the PI3K/AKT and the MEK/ERK pathways. Therefore, the inhibition of these pathways presents a potential therapeutic approach. In this study, we evaluated the efficacy of simultaneous inhibition of the PI3K/AKT and MEK/ERK pathways in docetaxel-resistant mCRPC, both in vitro and in vivo. Methods: Docetaxel-sensitive and docetaxel-resistant mCRPC cells were treated with selumetinib (MEK1/2 inhibitor), AZD8186 (PI3Kß/δ inhibitor) and capivasertib (pan-AKT inhibitor) alone and in combination. Efficacy and toxicity of selumetinib+AZD8186 were tested in docetaxel-resistant xenograft mice. CRISPR-Cas9 generated a PTEN-knockdown docetaxel-resistant cell model. Changes in phosphorylation of AKT, ERK and downstream targets were analyzed by Western blot. Antiapoptotic adaptations after treatments were detected by dynamic BH3 profiling. Results: PI3K/AKT and MEK/ERK pathways were hyperactivated in PTEN-wild-type (wt) docetaxel-resistant cells. Selumetinib+AZD8186 decreased cell proliferation and increased apoptosis in PTEN-wt docetaxel-resistant cells. This observation was further confirmed in vivo, where docetaxel-resistant xenograft mice treated with selumetinib+AZD8186 exhibited reduced tumor growth without additional toxicity. Conclusion: Our findings on the activity of selumetinib+AZD8186 in PTEN-wt cells and in docetaxel-resistant xenograft mice provide an excellent rationale for a novel therapeutic strategy for PTEN-wt mCRPC patients resistant to docetaxel, in whom, unlike PTEN-loss patients, a clinical benefit of treatment with single-agent PI3K and AKT inhibitors has not been demonstrated. A phase I-II trial of this promising combination is warranted.
ABSTRACT
Cytotoxic immune cells, including T lymphocytes (CTLs) and natural killer (NK) cells, are essential components of the host response against tumors. CTLs and NK cells secrete granzyme A (GzmA) upon recognition of cancer cells; however, there are very few tools that can detect physiological levels of active GzmA with high spatiotemporal resolution. Herein, we report the rational design of the near-infrared fluorogenic substrates for human GzmA and mouse GzmA. These activity-based probes display very high catalytic efficiency and selectivity over other granzymes, as shown in tissue lysates from wild-type and GzmA knock-out mice. Furthermore, we demonstrate that the probes can image how adaptive immune cells respond to antigen-driven recognition of cancer cells in real time.