Potent immunosuppressive mechanisms within the tumor microenvironment contribute to the resistance of aggressive human cancers to immune checkpoint blockade (ICB) therapy. One of the main mechanisms for myeloid-derived suppressor cells (MDSCs) to induce T cell tolerance is through secretion of reactive nitrogen species (RNS), which nitrates tyrosine residues in proteins involved in T cell function. However, so far very few nitrated proteins have been identified. Here, using a transgenic mouse model of prostate cancer and a syngeneic cell line model of lung cancer, we applied a nitroproteomic approach based on chemical derivation of 3-nitrotyrosine and identified that lymphocyte-specific protein tyrosine kinase (LCK), an initiating tyrosine kinase in the T cell receptor signaling cascade, is nitrated at Tyr394 by MDSCs. LCK nitration inhibits T cell activation, leading to reduced interleukin 2 (IL2) production and proliferation. In human T cells with defective endogenous LCK, wild type, but not nitrated LCK, rescues IL2 production. In the mouse model of castration-resistant prostate cancer (CRPC) by prostate-specific deletion of Pten, p53, and Smad4, CRPC is resistant to an ICB therapy composed of antiprogrammed cell death 1 (PD1) and anticytotoxic–T lymphocyte-associated protein 4 (CTLA4) antibodies. However, we showed that ICB elicits strong anti-CRPC efficacy when combined with an RNS neutralizing agent. Together, these data identify a previously unknown mechanism of T cell inactivation by MDSC-induced protein nitration and illuminate a clinical path hypothesis for combining ICB with RNS-reducing agents in the treatment of CRPC. Significance Immune checkpoint blockade (ICB) to reinvigorate cytotoxic T lymphocytes using antibodies against CTLA4 or PD1 generates durable therapeutic responses in patients across a variety of cancer types. However, some cancers such as castration-resistant prostate cancer (CRPC) show overwhelming resistance to ICB. Here, we develop a nitroproteomic approach and uncover a potential mechanism for the immunotherapy resistance where a key protein for T cell activation, lymphocyte-specific protein tyrosine kinase (LCK), is nitrated and inactivated by myeloid-derived suppressor cell–generated reactive nitrogen species (RNS) in the resistant tumor. An agent that neutralizes RNS significantly sensitizes CRPC to ICB therapy in a mouse model. Therefore, our studies illuminate a clinical path hypothesis for combining ICB with RNS-reducing agents in the treatment of ICB-resistant cancers.

Targeting and exploitation of tumor-associated neutrophils to enhance immunotherapy and drug delivery for cancer treatment

Resistance to immune checkpoint blockade (ICB) therapy represents a formidable clinical challenge limiting the efficacy of immunotherapy. In particular, prostate cancer poses a challenge for ICB therapy due to its immunosuppressive features. A ketogenic diet (KD) has been reported to enhance response to ICB therapy in some other cancer models. However, adverse effects associated with continuous KD were also observed, demanding better mechanistic understanding and optimized regimens for using KD as an immunotherapy sensitizer. In this study, we established a series of ICB-resistant prostate cancer cell lines and developed a highly effective strategy of combining anti-PD1 and anti-CTLA4 antibodies with histone deacetylase inhibitor (HDACi) vorinostat, a cyclic KD (CKD), or dietary supplementation of the ketone body β-hydroxybutyrate (BHB), which is an endogenous HDACi. CKD and BHB supplementation each delayed prostate cancer tumor growth as monotherapy, and both BHB and adaptive immunity were required for the antitumor activity of CKD. Single-cell transcriptomic and proteomic profiling revealed that HDACi and ketogenesis enhanced ICB efficacy through both cancer cell–intrinsic mechanisms, including upregulation of MHC class I molecules, and -extrinsic mechanisms, such as CD8+ T-cell chemoattraction, M1/M2 macrophage rebalancing, monocyte differentiation toward antigen-presenting cells, and diminished neutrophil infiltration. Overall, these findings illuminate a potential clinical path of using HDACi and optimized KD regimens to enhance ICB therapy for prostate cancer. Significance: Optimized cyclic ketogenic diet and 1,3-butanediol supplementation regimens enhance the efficacy of immune checkpoint blockade in prostate cancer through epigenetic and immune modulations, providing dietary interventions to sensitize tumors to immunotherapy.

Immunocyte infiltration and cytotoxicity play critical roles in both inflammation and immunotherapy. However, current cancer immunotherapy screening methods overlook the capacity of the T cells to penetrate the tumor stroma, thereby significantly limiting the development of effective treatments for solid tumors. Here, we present an automated high-throughput microfluidic platform for simultaneous tracking of the dynamics of T cell infiltration and cytotoxicity within the 3D tumor cultures with a tunable stromal makeup. By recourse to a clinical tumor-infiltrating lymphocyte (TIL) score analyzer, which is based on a clinical data-driven deep learning method, our platform can evaluate the efficacy of each treatment based on the scoring of T cell infiltration patterns. By screening a drug library using this technology, we identified an epigenetic drug (lysine-specific histone demethylase 1 inhibitor, LSD1i) that effectively promoted T cell tumor infiltration and enhanced treatment efficacy in combination with an immune checkpoint inhibitor (anti-PD1) in vivo. We demonstrated an automated system and strategy for screening immunocyte-solid tumor interactions, enabling the discovery of immuno- and combination therapies.