Photodynamic therapy (PDT) is an effective anticancer modality approved by the U.S. Food and Drug Administration (FDA). Antitumor immunity can be augmented during PDT by inducing sterile inflammation in an acute manner, and this process is characterized by interleukin 17 (IL‐17)‐mediated neutrophil infiltration to tumor‐draining lymph nodes (TDLNs). However, the inflammatory factors that influence IL‐17 expression in TDLNs are poorly understood. Prior studies have linked the cyclooxygenase 2 (COX2)‐driven prostaglandin E 2 (PGE2) pathway to IL‐17 expression. Here, we report that an immune‐activating PDT regimen (imPDT) induces COX2/PGE2 expression in TDLNs, whereby IL‐17 expression is facilitated without corresponding effects on the expression of RORγt, the transcriptional driver of the canonical IL‐17 pathway. Pharmacologic inhibition with NS398, a COX2 inhibitor, was utilized to demonstrate that imPDT‐induced COX2 regulates RORγt‐independent expression of IL‐17 by B cells and neutrophil entry into TDLNs. Depletion of B cells prior to imPDT significantly reduced neutrophil entry into TDLNs following treatment, and diminishes the efficacy of imPDT, which is dependent upon antitumor immunity. These findings are suggestive of a novel role for B cells in the augmentation of antitumor immunity by imPDT.

Objective: Patients with inoperable extrabronchial or endobronchial tumors who are not candidates for curative radiotherapy have dire prognoses with no effective long-term treatment options. To reveal that our computer-optimized interstitial photodynamic therapy (I-PDT) is safe and potentially effective in the treatment of patients with inoperable extra or endobronchial malignancies inducing central airway obstructions. Methods: High-spatial resolution computer simulations were used to personalize the light dose rate and dose for each tumor. Endobronchial ultrasound with a transbronchial needle was used to place the optical fibers within the tumor according to an individualized plan. The primary and secondary end points were safety and overall survival, respectively. An exploratory end point evaluated changes in immune markers. Results: Eight patients received I-PDT with planning, and five of these received additional external beam PDT. Two additional patients received external beam PDT. The treatment was declared safe. Three of 10 patients are alive at 26.3, 12, and 8.3 months, respectively, after I-PDT. The treatments were able to deliver a prescribed light dose rate and dose to 87% to 100% and 18% to 92% of the tumor volumes, respectively. A marked increase in the proportion of monocytic myeloid-derived suppressor cells expressing programmed death-ligand 1 was measured in four of seven patients. Conclusions: Image-guided light dosimetry for I-PDT with linear endobronchial ultrasound transbronchial needle is safe and potentially beneficial in increasing overall survival of patients. I-PDT has a positive effect on the immune response including an increase in the proportion of programmed death-ligand 1–expressing monocytic myeloid-derived suppressor cells.

Abstract Androgen receptor (AR) antagonism increases overall survival in prostate cancer; however, treatment failure leads to tumor progression and patient mortality. The effect of AR modulation on AR+ nontumor cells that participate in the resistance to AR antagonism is poorly understood. Tumor-infiltrating myeloid cells, including macrophages and myeloid-derived suppressor cells (MDSC), express AR and promote prostate cancer progression. We investigated how AR antagonism affects myeloid cell function and metabolism in an AR-independent murine colon tumor model. Systemic blockade of AR with enzalutamide resulted in increased MC-38 tumor growth in vivo even when AR was knocked out of MC-38 tumor cells. MC-38 tumor growth was also increased when immunocompetent, but not immunodeficient, mice were coinjected with tumor cells and MDSCs treated with enzalutamide or lacking AR, suggesting that AR regulated the ability of MDSCs to suppress adaptive immunity. Myeloid AR-knockout male mice also displayed increased growth of TRAMP C2 prostate tumors when compared with wild type. Inhibition of AR signaling suppressed mitochondrial respiration in myeloid cells via MPC/AMPK signaling pathways; suppression of mitochondrial respiration increased MDSC tumor–promoting functions. Our work showed that AR regulates a tumor-promoting myeloid cell phenotype and influences myeloid cell metabolism. These findings suggest that tumor resistance to AR antagonism is due, in part, to changes in myeloid cell function and metabolism.

Myeloid cells are critical cells involved in the orchestration of innate and adaptive immune responses. Most myeloid cells derive from the adult bone marrow in a process called myelopoiesis, a tightly controlled process that ensures constant production of myeloid cells. Sex differences in myeloid cell development have been observed; males exhibit greater monocytic differentiation in the bone marrow, and men have increased blood monocyte numbers when compared to women. Here we use a genetic mouse model of myeloid androgen receptor (AR) knockout (MARKO) and pharmacological inhibition of AR to investigate the role of androgen signaling in monocytic differentiation. We observe that although myeloid AR signaling does not influence total bone marrow cell numbers, it does affect the composition of the bone marrow myeloid population in both homeostatic and emergency settings. Genetic deletion of AR in myeloid cells led to reduced monocytic development in vivo. Similarly, pharmacologic inhibition of AR signaling in vitro reduced monocytic development. However, alteration in monocytic differentiation in the absence of AR signaling did not lead to reduced numbers of circulating myeloid cells, although MARKO male mice display reduced ratio of classical to non-classical monocytes in the blood, implying that blood monocyte subsets are skewed upon myeloid AR deletion. Our results suggest that the sex differences observed in monocytic differentiation are partly attributed to the positive role of the androgen-AR axis in regulating monocytic development directly at the myeloid cell level. Furthermore, we have identified a novel role for AR in regulating blood mature monocyte subset turnover. Investigating how androgen signaling affects monocytic development and monocyte subset heterogeneity will advance our understanding of sex differences in monocytic function at homeostasis and disease and can ultimately impact future therapeutic design targeting monocytes in the clinic.

It has been recently recognized that sex hormones can regulate hematopoiesis and modulate immune phenotypes that underlie sex disparities in immune response. Androgen receptor (AR) antagonism is known to compromise T cell response; however, the role of AR signaling in myeloid cell biology is poorly understood. To address this question, we analyzed the effect of AR blockage on functional activity of myeloid cells in MARKO (Myeloid AR KnockOut) mice and after pharmacological inhibition of AR. We demonstrated that AR antagonism promoted VEGFa, Arg1 and PD-L1 expression in MDSCs, and enhanced their immunosuppressive activity. AR blockage induced metabolic switch in myeloid cells by suppression of mitochondrial respiration and upregulation of glycolysis. AR-mediated metabolic reprogramming was regulated in a MPC/AMPK-dependent manner. To understand the role of AR antagonism in anti-tumor immunity, we utilized the AR-independent models of MC-38 colon and TRAMP C2 prostate carcinomas. Neither AR inhibition nor AR deletion in myeloid cells affected tumor infiltration with TAMs, MDSCs or T cells. However, in both models AR blockade and genetic deletion promoted tumor growth in immunocompetent but not immunodeficient mice, suggesting an important role of AR-mediated regulation of immunosuppression in anti-tumor immunity. Taken together, our findings reveal that AR signaling represents an important regulator of myeloid cell functional activity, and suggest a novel tumor-extrinsic mechanism of prostate cancer relapse.

Photodynamic therapy (PDT) is an FDA-approved modality for the treatment of early-stage disease and palliation of late-stage disease. Pre-clinical studies using mouse models and clinical studies in patients have demonstrated that PDT is capable of influencing the immune system. The effect of PDT on the generation of anti-tumor immunity is regimen-dependent and is tightly linked to the degree and nature of inflammation induced by PDT. However, the precise mechanism underlying PDT-regulated adaptive anti-tumor immunity remains unclear. This review will focus on the current knowledge of immune regulation by PDT.

Purpose: The aim of this study is to investigate whether radiofrequency ablation (RFA) improves the efficacy of adoptive T cell immunotherapy in preclinical mouse cancer models. Method: Mice implanted subcutaneously (sc) with syngeneic colon adenocarcinoma or melanoma were treated with sub-curative in situ RFA (90 °C, 1 min). Trafficking of T cells to lymph nodes (LN) or tumors was quantified by homing assays and intravital microscopy (IVM) after sham procedure or RFA. Expression of trafficking molecules (CCL21 and intercellular adhesion molecule-1 [ICAM-1]) on high endothelial venules (HEV) in LN and tumor vessels was evaluated by immunofluorescence microscopy. Tumor-bearing mice were pretreated with RFA to investigate the therapeutic benefit when combined with adoptive transfer of in vitro-activated tumor-specific CD8+ T cells. Results: RFA increased trafficking of naïve CD8+ T cells to tumor-draining LN (TdLN). A corresponding increase in expression of ICAM-1 and CCL21 was detected on HEV in TdLN but not in contralateral (c)LN. IVM revealed that RFA substantially enhanced secondary firm arrest of lymphocytes selectively in HEV in TdLN. Furthermore, strong induction of ICAM-1 in tumor vessels was associated with significantly augmented trafficking of adoptively transferred in vitro-activated CD8+ T cells to tumors after RFA. Finally, preconditioning tumors with RFA augmented CD8+ T cell-mediated apoptosis of tumor targets and delayed growth of established tumors when combined with adoptive T cell transfer immunotherapy. Conclusions: These studies suggest that in addition to its role as a palliative therapeutic modality, RFA may have clinical potential as an immune-adjuvant therapy by augmenting the efficacy of adoptive T cell therapy.

In addition to direct killing of tumor cells, PDT can enhance anti-tumor immune responses in the host. It is well known that PDT-induced acute inflammation facilitates PDT-enhanced anti-tumor immunity. Our studies in murine colon carcinoma model demonstrates that PDT induces a short-lived burst of PGE2 in tumor draining lymph node which is critical for ability of PDT to induce acute inflammation. Using a selective COX2 inhibitor NS398, we demonstrate that this short-lived PGE2 burst regulates PDT-enhanced anti-tumor immunity and overall PDT efficacy. These results bring to light a beneficial role of PDT-induced acute expression of PGE2 on PDT-enhanced anti-tumor immunity. Although long term administration of NSAIDS is the current clinical practice for PDT, our research emphasizes on delaying the timing of NSAID administration to after acute inflammation is resolved for optimal response.

Photodynamic therapy (PDT) is a non-invasive FDA and EMA-approved anticancer treatment modality. Initially developed for elimination of malignant cells, PDT affects all cells in the tumor bed including stromal cells. Stroma represents not only an important component of tumor microenvironment, but has a significant impact on tumor susceptibility to PDT and other anticancer therapies. However, the effects of PDT on stromal cells are poorly investigated. During PDT the tumor stroma can receive low-dose irradiation as a result of chosen regimen or limited depth of light penetration. Here, we characterized response of human mesenchymal stromal cells (MSCs) to low-dose PDT. In an in vitro model we demonstrated that low-dose PDT resulted in activation of Erk1/2 and inhibition of GSK-3 signaling in MSCs. PDT-mediated induction of intracellular reactive oxygen species (ROS) resulted in reorganization of MSC cytoskeleton and decreased cell motility. More importantly, low-dose PDT dramatically upregulated secretion of various proangiogenic factors (VEGF-A, IL-8, PAI-1, MMP-9, etc.) by MSCs and improved MSC ability to promote angiogenesis suggesting an increase in the pro-tumorigenic potential of MSCs. In contrast, co-cultivation of PDT-treated MSCs with lymphocytes resulted in significant decrease of MSC viability and potential increase in MSC immunogenicity, which may lead to increased anti-tumor immunity. Low-dose PDT in MSCs significantly inhibited secretion of CCL2 (MCP-1) potentially limiting infiltration of pro-tumorigenic macrophages. Altogether, our findings demonstrate that low-dose PDT significantly modifies functional properties of MSCs improving their pro-tumorigenic potential while simultaneously increasing potential immune stimulation suggesting possible mechanisms of stromal cell contribution to PDT efficacy.

The effect of PDT on the host immune response and the role the host immune response plays in PDT efficacy against cancer has been the subject of intensive research for the past 2 plus decades. During that time we have learned that 1) the patient’s immune health can affect the efficacy of PDT; 2) PDT can both enhance and suppression immunity; 3) treatment regimens can be devised to enhance anti-tumor immunity; and 4) PDT has the potential to be an effective immunotherapy. This presentation will focus on how these findings came about and the role Dr. Dougherty played in the discoveries.

IL-1 is a pro-inflammatory cytokine essential for host defense against pathogens, but its role in cancer progression remains understudied. Recently our lab identified the IL-1 pathway as a molecular mechanism responsible for myeloid-driven prostate cancer development. Here, we report that IL-1 signaling in tumor cells drives prostate cancer progression. To identify the contribution of IL-1 signaling in prostate cancer progression, we downregulated expression of IL1R1 (the only known functional receptor for IL-1α/β) using shRNA or Crisr/Cas9 in androgen-sensitive (LNCaP) and androgen-independent (PC-3M) prostate cancer cell lines. We found that IL-1α inhibited expression of androgen receptor in LNCaP while suppression of IL-1 signaling abrogated this effect. The expression of neuron-specific enolase which was shown to be a prognostic marker associated with poor survival outcome was significantly reduced in PC-3M cells lacking IL1R1. Downregulation of IL-1 signaling in cancer cells resulted in significant inhibition of prostate tumor growth while no effect on cell proliferation was detected. We established this phenomenon and found several possible mechanisms underlying it. We found that knocking down of IL1R1 in cancer cells resulted in suppression of mTOR and ACC signaling in tumors. TUNEL staining revealed increased number of apoptotic cells in shIL1R1-PC-3M tumors. Tumors lacking IL1R1 were characterized by impaired vascularization which was accompanied by reduced COX-2, VEGF-A and IL-8 expression. Taken together, our findings indicate that downregulation of IL-1 signaling modifies prostate cancer cell phenotype and suppresses tumor growth.

Over the past several decades hundreds of cancer patients have been treated with PDT in both clinical trials and off label. PDT has been used for a wide variety of malignancies, including lung, esophageal, head and neck and pancreatic cancer, as well as mesothelioma. Treatment regimens and photosensitizer usage has also been variable. The knowledge gained from these studies has helped PDT to move into acceptance within some areas of the medical community, but the progress has been slow. This is due in part to a lack of Phase II and III randomized clinical trials in which PDT is measured against the standard of care. In addition, the wide variety of treatment parameters and study protocols has made it difficult to draw general conclusions on the factors that affect the efficacy of PDT. The knowledge gained from these studies has helped PDT to move into acceptance within some areas of the medical community, but the progress has been slow. The Registry will be grouped by disease site; we have developed lung, esophageal and mesothelioma to date with plans to expand to other sites. Data collected within the registry will include patient characteristics, PDT procedure specifics, outcomes, complications and quality of life assessments. Data will be searchable and the registry will be set up to provide reports to specific inquiries. Registry participation is open to all clinicians and researchers. A demonstration of the registry and its attributes will be given during this talk.

The etiology of prostate cancer is poorly understood, but it is a multi-step process that has been linked to environmental factors that induce inflammation within the gland. Glands of prostate cancer patients frequently contain multiple zones of disease at various stages of progression. The factors that drive disease progression from an indolent benign stage to aggressive disease are not well-defined. Prostate inflammation and carcinoma are associated with high levels of myeloid cell infiltration; these cells are linked to disease progression in other cancers, but their role in prostate cancer is unclear. To determine whether myeloid cells contribute to prostate cancer progression, the ability of prostate tumor-associated CD11b+ cells (TAMC) to drive prostate epithelial cell tumorigenesis was tested. Co-culture of CD11b+ TAMC with non-tumorigenic genetically primed prostate epithelial cells resulted in stable transformation and induction of tumorigenesis. RNA sequencing identified the IL-1α pathway as a potential molecular mechanism responsible for tumor promotion by TAMC. Inhibition of IL-1α delayed growth of TAMC-induced tumors. Further analysis showed that IL-1α inhibition led to decreased angiogenesis within tumors, suggesting that IL-1α promotes prostate tumor progression, potentially through augmentation of angiogenesis.

Abstract Innate immune modulators can generate an antitumor T cell response. However, significant toxicities associated with systemic administration have significantly limited their clinical use. The natural TLR5 agonist flagellin is unique amonginnate immune modulators because the tissue specificity of TLR5 expression induces a uniquely safe profile of cytokines following systemic TLR5 activation. Entolimod, a pharmacologically optimized flagellin derivative, was initially developed to treat and prevent acute radiation syndrome. Systemic administration of entolimod also showed antitumor effects in multiple preclinical mouse models mimicking clinically occurring liver metastasis. Entolimod suppresses liver metastasis through activation of NF-κB-, AP-1-, and STAT-3-driven immunomodulatory pathways in hepatocytes and a highly coordinated CD4+ T cell independent NK-DC-CD8+ T cell response. Although these studies characterized entolimod as a liver tropic immunotherapy, entolimod also suppresses spontaneous mammary lung metastasis. In contrast to the liver metastatic model, CD4+ T cells are required for the antitumor NK and CD8+ T cell response, indicating that the etiology of the cancer may explain the difference in the therapeutic effects ofTLR5 agonists. These results further strengthen that TLR5 agonists are a safe, effective and broadly applicable immunotherapeutic agent against metastases, which are currently a major cause of cancer-associated mortality. Recent completion of a phase I trial of entolimod in patients with advanced metastatic solid tumors has provided the rationale to test that the efficacy demonstrated in animal cancer models can be translated into immunotherapy of human tumors.

Compelling evidence has suggested the relevance of miRNAs in resistance to chemotherapeutic agents in HCC. miR-505 was reported to be downregulated and function as a tumor suppressor in HCC cells by binding to high-mobility group box 1 (HMGB1). Whether miR-505/HMGB1 axis was involved in ADM cytotoxicity in HCC remains to be addressed. The aim of this study was to explore the effect of miR-505/HMGB1 axis on ADM cytotoxicity in HCC cells. MTT, flow cytometry analysis, and caspase-3 activity assays were conducted to assess ADM-induced cytotoxicity. The protein level of phosphorylation of histone H2 AX at Ser139 (γH2AX) was detected to evaluate DNA damage. The effects of miR-505 and HMGB1 on the protein kinase B (Akt) pathway were determined by examining the protein levels of phosphorylated Akt (p-Akt), Akt, phosphorylated glycogen synthase kinase-3β (p-GSK-3β), and GSK-3β. We found that HMGB1 knockdown and miR-505 overexpression exacerbated ADM-induced cell viability inhibition, enhanced ADM-induced apoptosis, and increased caspase-3 activity in ADM-treated HCC cells. However, HMGB1 overexpression reversed the effects of miR-505 on ADM-induced cytotoxicity in HCC cells. HMGB1 knockdown and miR-505 overexpression promoted ADM-induced DNA damage in HCC cells, which was abated by HMGB1 overexpression. On a molecular mechanism level, HMGB1 silencing and miR-505 overexpression inactivated the Akt pathway in HCC cells, while exogenous HMGB1 resisted miR-505-induced Akt pathway inactivation. In conclusion, miR-505 overexpression enhanced ADM-induced cytotoxicity in HCC cells, at least partly by targeting HMGB1 and inactivating the Akt pathway.

Constitutive p16Ink4a expression, along with senescence-associated β-galactosidase (SAβG), are commonly accepted biomarkers of senescent cells (SCs). Recent reports attributed improvement of the healthspan of aged mice following p16Ink4a-positive cell killing to the eradication of accumulated SCs. However, detection of p16Ink4a/SAβG-positive macrophages in the adipose tissue of old mice and in the peritoneal cavity of young animals following injection of alginate-encapsulated SCs has raised concerns about the exclusivity of these markers for SCs. Here we report that expression of p16Ink4a and SAβG in macrophages is acquired as part of a physiological response to immune stimuli rather than through senescence, consistent with reports that p16Ink4a plays a role in macrophage polarization and response. Unlike SCs, p16Ink4a/SAβG-positive macrophages can be induced in p53-null mice. Macrophages, but not mesenchymal SCs, lose both markers in response to M1- [LPS, IFN-α, Poly(I:C)] and increase their expression in response to M2-inducing stimuli (IL-4, IL-13). Moreover, interferon-inducing agent Poly(I:C) dramatically reduced p16Ink4a expression in vivo in our alginate bead model and in the adipose tissue of aged mice. These observations suggest that the antiaging effects following eradication of p16Ink4a-positive cells may not be solely attributed to SCs but also to non-senescent p16Ink4a/SAβG-positive macrophages.

According to the CDC prostate cancer (CaP) has the highest incidence and second highest mortality rate amongst cancers in American men. Constitutive NF-κB activation is a hallmark of CaP and this pathway drives many pro-tumorigenic characteristics of CaP cells, including cell proliferation and survival. An activated NF-κB gene signature is predictive of CaP progression and biochemical recurrence following therapeutic intervention. However, the mechanisms that perpetuate NF-κB activation are incompletely understood. Genes that control NF-κB activity are rarely mutated in CaP suggesting that epigenetic mechanisms may contribute to constitutive NF-κB activation. microRNAs (miRs) epigenetically regulate many genes involved with NF-κB activation. IκBα is a direct inhibitor of NF-κB; it binds to and sequesters NF-κB in the cytoplasm resulting in functional inhibition. IκBα is a target gene of miR-30e* yet the expression and oncological impact of miR-30e* in CaP is unknown. We report that miR-30e* expression is elevated in multiple murine models of CaP and is most pronounced in late stage disease. miR-30e* drives CaP proliferation and tumor growth through inhibition of IκBα, which results in chronic activation of NF-κB. Additionally, we show that inhibition of miR-30e* improves chemotherapeutic control of CaP. Thus, miR-30e* may prove to be a novel clinical target whose inhibition leads to decreased CaP cell proliferation and sensitization of CaP cells to chemotherapeutics.

Activation of an anticancer innate immune response is an attractive immunotherapeutic opportunity that is challenged by insufficient safety of systemically administered innate immune modulators. Unusual tissue specificity of expression of TLR5 determines a uniquely safe profile of cytokines induced by the sole TLR5 agonist flagellin. Entolimod, a pharmacologically optimized flagellin derivative, was initially developed to treat and prevent acute radiation syndrome with demonstrated efficacy in rodents and non-human primates and safety in human healthy volunteers. In addition, Entolimod demonstrated antitumor effects in mouse models of uveal melanoma, lymphoma, breast, and colorectal carcinoma. Entolimod’s mechanism of action involves activation of NF-κB-, AP-1-, and STAT-3-driven immunomodulatory pathways in hepatocytes that initiates a cascade of cell-cell signaling events that mobilize innate and adaptive immunity to the liver. This includes CXCR3-dependent blood-borne NK cell homing followed by DC activation and antitumor CD8+ T cell memory formation. These results define systemically administered TLR5 agonists as an organ-specific immunoadjuvant enabling efficient antitumor vaccination that does not depend on identification of tumor-specific antigens. Therefore, Entolimod has strong promise as a safe, effective and broadly applicable immunotherapeutic agent against liver metastases, which are currently a major cause of cancer-associated mortality. Recent completion of a phase I trial of Entolimod in patients with advanced metastatic solid tumors has opened the opportunity to test whether efficacy demonstrated in animal cancer models could be translated into immunotherapy of human tumors.

Difficulties differentiating indolent from progressing prostate cancer (CaP) results in ineffective treatment strategies. Tumor-associated myeloid cells (TAMC) contribute to tumor progression; yet the role TAMC play in the conversion from indolent to progressing CaP is unclear. To test whether TAMC promote CaP progression we developed a murine model using two isogenic cell lines from the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model. Tumorigenic C2 cells mimic progressing disease while non-tumorigenic C3 cells represent indolent disease. We report for the first time that CD11b+ TAMC isolated from C2 tumors co-injected with the non-tumorigenic C3 cells drive C3 tumor growth. In contrast, TAMC isolated from C2 tumor-bearing spleens or naïve spleens did not promote C3 tumor growth. This is the first indication that TAMC drive progression of indolent disease. Strikingly, tumor cells explanted from TAMC-induced C3 tumors were tumorigenic in the absence of additional TAMC, suggesting that TAMC induce stable changes within the C3 cells. These results have been recapitulated with the non-tumorigenic human cell line BPH-1 (benign prostatic hyperplasia). Furthermore, TAMC-induced tumor growth occurs via a TAMC secreted factor. Using a 5-day transwell system PC3M (human prostatic adenocarcinoma) TAMC were able to drive BPH-1 tumorigenesis. Future work will determine the factor secreted by TAMC and identify the molecular mechanisms responsible for tumor promotion. This work has the potential to identify critical factors responsible for the conversion of indolent to progressing disease thus laying the groundwork for novel prognostic and therapeutic strategies.