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Introduction: Immune checkpoint blockade (ICB) improved clinical outcomes in renal and bladder cancer patients, but the response rates remain limited especially in metastatic disease. While STAT3 transcription factor is well-known master regulator of tumor immune evasion, little is known about the role of STAT3 in the resistance of renal or bladder cancers to immunotherapy. Methods: To better understand immune alterations associated with ICB resistance, we assessed blood biomarkers in renal cancer patients classified as responders or non-responders to first line nivolumab/ipilimumab immunotherapy. Results: We observed that non-responders showed elevated levels of proinflammatory mediators, such as IL-1RA, IL-6, IL-8 and to lesser extent IL-10, which are associated with STAT3 activation and tumor immunosuppression. In addition, we found STAT3 activation primarily in circulating myeloid immune cells such as tolerogenic MDSCs. To assess whether STAT3 inhibition within these cell subsets can promote antitumor immune responses and/or enhance sensitivity to ICB in vivo, we used an original antisense oligonucleotide (ASO) strategy for myeloid-cell selective STAT3 knockdown (CpG-STAT3ASO). Our results in syngeneic models of renal and bladder cancers in mice demonstrated potent antitumor activity of CpG-STAT3ASO alone in contrast to PD1 blockade alone in both models. The CpG-STAT3ASO/anti-PD1 combination improved therapeutic efficacy especially against bladder tumors. Therapeutic efficacy correlated with activation of dendritic cells (DCs) and M1 macrophages in the tumor microenvironment, reduced percentages of regulatory T cells (Tregs) and the expansion of CD8 T cells in both tumor models. Discussion/Conclusion: Our study underscores the potential of using myeloidcell targeted CpG-STAT3 inhibitors for genitourinary cancer therapy to disrupt tolerogenic signaling, restore immune cell activity and sensitivity to immune checkpoint inhibitors and/or T cell-based immunotherapies. [ABSTRACT FROM AUTHOR]

Cell-selective delivery and sensitivity to serum nucleases remain major hurdles to the clinical application of RNA-based oligonucleotide therapeutics, such as siRNA. Spider silk shows great potential as a biomaterial due to its biocompatibility and biodegradability. Self-assembling properties of silk proteins allow for processing into several different morphologies such as fibers, scaffolds, films, hydrogels, capsules and spheres. Moreover, bioengineering of spider silk protein sequences can functionalize silk by adding peptide moieties with specific features including binding or cell recognition domains. We demonstrated that modification of silk protein by adding the nucleic acid binding domain enabled the development of a novel oligonucleotide delivery system that can be utilized to improve pharmacokinetics of RNA-based therapeutics, such as CpG-siRNA. The MS2 bioengineered silk was functionalized with poly-lysine domain (KN) to generate hybrid silk MS2KN. CpG-siRNA efficiently bound to MS2KN in contrary to control MS2. Both MS2KN complexes and spheres protected CpG-siRNA from degradation by serum nucleases. CpG-siRNA molecules encapsulated into MS2KN spheres were efficiently internalized and processed by TLR9-positive macrophages. Importantly, CpG- STAT3 siRNA loaded in silk spheres showed delayed and extended target gene silencing compared to naked oligonucleotides. The prolonged Stat3 silencing resulted in the more pronounced downregulation of interleukin 6 (IL-6), a proinflammatory cytokine and upstream activator of STAT3, which limits the efficacy of TLR9 immunostimulation. Our results demonstrate the feasibility of using spider silk spheres as a carrier of therapeutic nucleic acids. Moreover, the modified kinetic and activity of the CpG- STAT3 siRNA embedded into silk spheres is likely to improve immunotherapeutic effects in vivo . Statement of Significance We demonstrated that modification of silk protein by adding the nucleic acid binding domain enabled the development of a novel oligonucleotide delivery system that can be utilized to improve pharmacokinetics of RNA-based therapeutics. Although, the siRNA constructs have already given very promising results in the cancer therapy, the in vivo application of RNA-based oligonucleotide therapeutics still is limited due to their sensitivity to serum nucleases and some toxicity. We propose a carrier for RNA-based therapeutics that is made of bioengineered spider silk. We showed that functionalized bioengineered spider silk spheres not only protected RNA-based therapeutics from degradation by serum nucleases, but what is more important the embedding of siRNA into silk spheres delayed and extended target gene silencing compared with naked oligonucleotides. Moreover, we showed that plain silk spheres did not have unspecific effect on target gene levels proving not only to be non-cytotoxic but also very neutral vehicles in terms of TLR9/STAT3 activation in macrophages. We demonstrated advantages of novel delivery technology in safety and efficacy comparing with delivery of naked CpG- STAT3 siRNA therapeutics. [ABSTRACT FROM AUTHOR]

TLR9 upregulation in prostate cancer cells results in the LIF‐mediated recruitment and increased tolerogenic activity of polymorphonuclear myeloid‐derived suppressor cells. Proinflammatory signals promote prostate tumorigenesis and progression, but their origins and downstream effects remain unclear. We recently demonstrated that the expression of an innate immune receptor, TLR9, by prostate cancer cells is critical for their tumor‐propagating potential. We investigated whether cancer cell–intrinsic TLR9 signaling alters composition of the prostate tumor microenvironment. We generated Ras/Myc (RM9) and Myc‐driven (Myc‐CaP) prostate cancer cells expressing the tetracycline‐inducible gene Tlr9 (Tlr9ON) or the control LacZ (LacZON). When engrafted into mice and treated with tetracycline, Tlr9ON, but not LacZON, tumors showed accelerated growth kinetics compared with tumors in PBS‐treated mice. Tlr9 upregulation in cancer cells triggered the selective accumulation of CD11b+Ly6GHILy6CLO myeloid cells, phenotypically similar to PMN‐MDSCs. The PMN‐MDSCs from tetracycline‐treated RM9‐Tlr9ON tumors increased the immunosuppressive activity of the STAT3 transcription factor, thereby more potently inhibiting T cell proliferation. We identified LIF, an IL‐6‐type cytokine and STAT3 activator, as a potential mediator of crosstalk between TLR9‐expressing prostate cancer cells and PMN‐MDSCs. Antibody‐mediated LIF neutralization reduced the percentage of tumor‐infiltrating PMN‐MDSCs and inhibited tumor growth in mice. The clinical relevance of LIF is confirmed by the correlation between TLR9 and LIF expression in prostate cancer specimens. Furthermore, blood samples from patients with prostate cancer showed elevated levels of LIF and high LIFR expression on circulating PMN‐MDSCs. Our results suggest that TLR9+ prostate cancers promote immune evasion via LIF‐mediated expansion and activation of PMN‐MDSCs. Finally, targeting TLR9/LIF/STAT3 signaling using oligonucleotide‐based inhibitors, such as CpG‐STAT3dODN, can offer new opportunities for prostate cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Simple Summary: Following local treatment of prostate cancer by surgical removal or radiation, biochemical recurrence may occur and progress to castration resistance (CR) following androgen deprivation therapy (ADT). If disease persists, men develop metastatic disease (mCRPC) which leads to death. Prior to mCRPC, a non-metastatic state exists (nmCRPC) characterized by a rise in PSA and lack of detectable metastases. Here, we review potential therapeutic strategies to interfere with the transition before the cancer becomes deadly. Nearly one third of men will incur biochemical recurrence after treatment for localized prostate cancer. Androgen deprivation therapy (ADT) is the therapeutic mainstay; however, some patients will transition to a castrate resistant state (castrate resistant prostate cancer, CRPC). Subjects with CRPC may develop symptomatic metastatic disease (mCRPC) and incur mortality several years later. Prior to metastatic disease, however, men acquire non-metastatic CRPC (nmCRPC) which lends the unique opportunity for intervention to delay disease progression and symptoms. This review addresses current therapies for nmCRPC, as well as novel therapeutics and pathway strategies targeting men with nmCRPC. [ABSTRACT FROM AUTHOR]

Myeloid immune cells, such as dendritic cells, monocytes, and macrophages, play a central role in the generation of immune responses and thus are often either disabled or even hijacked by tumors. These new tolerogenic activities of tumor-associated myeloid cells are controlled by an oncogenic transcription factor, signal transducer and activator of transcription 3 (STAT3). STAT3 multitasks to ensure tumors escape immune detection by impairing antigen presentation and reducing production of immunostimulatory molecules while augmenting the release of tolerogenic mediators, thereby reducing innate and adaptive antitumor immunity. Tumor-associated myeloid cells and STAT3 signaling in this compartment are now commonly recognized as an attractive cellular target for improving efficacy of standard therapies and immunotherapies. Hereby, we review the importance and functional complexity of STAT3 signaling in this immune cell compartment as well as potential strategies for cancer therapy. [ABSTRACT FROM AUTHOR]

The myeloid-derived suppressor cells (MDSCs) contribute to tumor immune evasion and still remain an elusive therapeutic target. Our study identified granulocytic MDSCs accumulating in prostate cancer patients during disease progression. We demonstrate the feasibility of using STAT3siRNA-based strategy for targeting MDSCs to alleviate arginase-dependent suppression of T cell activity. [ABSTRACT FROM AUTHOR]