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73 Background: 223Ra and 177Lu-PSMA-617 both prolong overall survival (OS) in different mCRPC settings. The observational, retrospective study, RALU, investigated safety and clinical outcomes of sequential 223Ra/177Lu-PSMA therapy in patients (pts) with mCRPC. This analysis evaluated the association of time interval between 223Ra and 177Lu-PSMA treatments and safety and OS outcomes of 177Lu-PSMA. Methods: Retrospective data were collected from 2021–22 in German nuclear medicine centers for all pts receiving 177Lu-PSMA with prior history of 223Ra therapy. Time intervals were 223Ra dose to first 177Lu-PSMA dose. Results: 42 pts received 177Lu-PSMA within 6 mo after 223Ra (Grp 1) and 90 pts received 223Ra ≥6 mo prior to 177Lu-PSMA (Grp 2). Baseline characteristics prior to 177Lu-PSMA therapy were, respectively: median ages 72 and 74 years; 57% and 63% with Eastern Cooperative Oncology Group performance status (ECOG PS) 1, 43% and 37% with ECOG PS 2; median prostate-specific antigen (PSA) values were 366 and 268 ng/ml, and median alkaline phosphatase (ALP) values were 133 and 149 U/L; 40% and 64% received ≥4 life prolonging therapies before starting 177Lu-PSMA. All pts had prior 223Ra; 57% and 77% received 6 223Ra injections; other prior therapies were abiraterone (60%, 77%), enzalutamide (50%, 78%), docetaxel (71%, 76%) and cabazitaxel (17%, 26%). Prior to 177Lu-PSMA, 24% and 29% of pts had visceral metastases. 45% and 52% of pts received ≥4 177Lu-PSMA cycles. From 177Lu-PSMA start to ≤30 days post last dose, 71% and 82% of pts had treatment-emergent adverse events (TEAEs) of any grade; most common were fatigue (12%, 7%), nausea (12%, 8%) and dry mouth (7%, 18%); 36% and 24% of pts had grade 3–4 TEAEs; excluding laboratory abnormalities, osteonecrosis of the jaw was the most frequent grade 3–4 TEAE (5%, 2%). Grade 3–4 laboratory abnormalities (177Lu-PSMA start to ≤90 days post last dose) are shown; treatment-related deaths were reported for 2% and 4% of pts. AEs led to treatment delays in 10% and 9% of pts. Median OS from start of 177Lu-PSMA was 12.0 mo (95% CI, 8.8–19.9) in Grp 1 and 13.2 mo (95% CI, 10.0–15.9) in Grp 2. During 177Lu-PSMA therapy, PSA response ≥50% occurred in 53% and 39% and ALP response ≥30% in 28% and 14% of pts, respectively. Conclusions: In this real-world setting, treating pts with 177Lu-PSMA within 6 mo of completing 223Ra was clinically feasible and well tolerated: no safety signals or concerns were seen. OS outcomes were similar in pts receiving 177Lu-PSMA 223Ra. [Table: see text]

5044 Background: Darolutamide (DARO), a highly potent and structurally distinct androgen receptor inhibitor, prolonged metastasis-free survival by nearly 2 years and reduced the risk of death by 31% vs placebo (PBO) with a favorable tolerability profile in patients (pts) with nonmetastatic castration-resistant prostate cancer (nmCRPC) in ARAMIS. We present post-hoc analyses of ARAMIS to evaluate the association between metastatic progression with prostate-specific antigen (PSA) and clinical progression and to describe the distribution of metastatic progression between groups. Methods: Pts with nmCRPC were randomized 2:1 to DARO (n=955) or PBO (n=554) while continuing androgen-deprivation therapy. Descriptive analyses were performed using the primary data cutoff (Sept 3, 2018) for the double-blind period. Post-baseline metastases were based on central review of conventional radiographic imaging every 16 weeks. PSA and pain progression were defined per primary analysis ( N Engl J Med. 2019;380:1235-46). Results: Metastatic progression was observed in 13.6% of DARO and 28.5% of PBO pts. Most pts had isolated progression as bone (DARO 46%, PBO 39%) or lymph node (32%; 40%) metastasis (Table). Pts with radiographic progression had shorter median time from initial diagnosis to study treatment (DARO 72.9, PBO 74.4 months) vs the overall ARAMIS population (86.2, 84.2 months). Of all pts with metastatic progression, baseline PSA levels (ng/mL) were similar in DARO (12.6) and PBO pts (15.1); DARO pts had lower median PSA before metastasis (16.7) vs PBO pts (48.0) and median absolute/relative PSA decrease from baseline of -0.7/-3.2% vs an increase for PBO pts of 29.5/181%. PSA progression before metastasis was observed in 55.6% (160/288) of pts, occurring in fewer DARO (45.4%) vs PBO pts (63.9%) (treatment difference 18.5%; nominal 95% CI 6.5%–30.6%). The median time between PSA progression and metastasis was 7.0 months with DARO vs 5.6 months with PBO. Pain progression before metastatic progression was rare and similar between groups (DARO 16.9%, PBO 17.7%). Conclusions: DARO significantly reduced risk of metastatic progression and improved overall survival vs PBO without changing the pattern of metastatic progression. Many pts with nmCRPC experienced metastatic progression without PSA progression, and pain progression was rare. These results support the use of imaging with PSA monitoring to properly identify disease progression in pts with nmCRPC. Clinical trial information: TBC. [Table: see text]

Immune microenvironment plays a critical role in lung cancer control versus progression and metastasis. In this investigation, we explored the effect of tumor-infiltrating lymphocyte subpopulations on lung cancer biology by studying in vitro cocultures, in vivo mouse models, and human lung cancer tissue. Lymphocyte conditioned media (CM) induced epithelial-mesenchymal transition (EMT) and migration in both primary human lung cancer cells and cell lines. Correspondingly, major accumulation of Th9 and Th17 cells was detected in human lung cancer tissue and correlated with poor survival. Coculturing lung cancer cells with Th9/Th17 cells or exposing them to the respective CM induced EMT in cancer cells and modulated the expression profile of genes implicated in EMT and metastasis. These features were reproduced by the signatory cytokines IL-9 and IL-17, with gene regulatory profiles evoked by these cytokines partly overlapping and partly complementary. Coinjection of Th9/Th17 cells with tumor cells in WT, Rag1-/-, Il9r-/-, and Il17ra-/- mice altered tumor growth and metastasis. Accordingly, inhibition of IL-9 or IL-17 cytokines by neutralizing antibodies decreased EMT and slowed lung cancer progression and metastasis. In conclusion, Th9 and Th17 lymphocytes induce lung cancer cell EMT, thereby promoting migration and metastatic spreading and offering potentially novel therapeutic strategies.

Rationale Recent studies indicate that tumor-associated macrophages (MΦ) with an M2 phenotype can influence cancer progression and metastasis, but the regulatory pathways remain poorly characterized. Objectives This study investigated the role of tumor-associated MΦ in lung cancer. Methods Coculturing of MΦ with mouse Lewis lung carcinoma (LLC1) and 10 different human lung cancer cell lines (adenocarcinoma, squamous cell carcinoma, and large cell carcinoma) caused up-regulation of CCR2/CCL2 and CX3CR1/CX3CL1 in both the cancer cells and the MΦ. Measurements and main results In the MΦ-tumor cell system, IL-10 drove CCR2 and CX3CR1 up-regulation, whereas CCL1, granulocyte colony-stimulating factor, and MIP1α were required for the up-regulation of CCL2 and CX3CL1. Downstream phenotypic effects included enhanced LLC1 proliferation and migration and MΦ M2 polarization. In vivo, MΦ depletion (clodronate, MΦ Fas-induced apoptosis mice) and genetic ablation of CCR2 and CX3CR1 all inhibited LLC1 tumor growth and metastasis, shifted tumor-associated MΦ toward M1 polarization, suppressed tumor vessel growth, and enhanced survival (metastasis model). Furthermore, mice treated with CCR2 antagonist mimicked genetic ablation of CCR2, showing reduced tumor growth and metastasis. In human lung cancer samples, tumor MΦ infiltration and CCR2 expression correlated with tumor stage and metastasis. Conclusions Tumor-associated MΦ play a central role in lung cancer growth and metastasis, with bidirectional cross-talk between MΦ and cancer cells via CCR2 and CX3CR1 signaling as a central underlying mechanism. These findings suggest that the therapeutic strategy of blocking CCR2 and CX3CR1 may prove beneficial for halting lung cancer progression.

The tumor microenvironment and its immune components play a critical role in cancer development, progression, and control. In this study we aim to investigate the role of tumor infiltrating lymphocyte subpopulations in lung cancer progression. Conditioned media (CM) from co-cultures of human lymphocytes with adenocarcinoma cells (A549) induced the loss of the epithelial markers (E-Cadherin and ZO2 and an increase of mesenchymal markers (vimentin, N-cadherin on mRNA and protein level. In addition, the cells demonstrated a spindled shape-like morphological changes and an increased migratory property. In order to explore the molecular mechanism that led to lymphocyte-induced EMT and migration, we performed a cytokine array from the co-culture CM and found elevated levels of IL-8, IL-16, CCL2 and G-CSF. Furthermore, we observed that lymphocyte-induced EMT was mediated via a TGFβ-independent pathway that involves phosphorylation of ERK1/2. Notably, this EMT phenotype induction by lymphocytes was independent of the pre-activation of lymphocytes with PMA. Furthermore, in order to identify the specific CD4+ T cell subpopulations (Th0, Th1, Th9 and Th17) responsible for the EMT effect, we generated specific subpopulations from mouse spleen T cells. Interestingly, we observed that Th9 and Th17 subpopulation CM led to EMT and increased migratory phenotype in mouse lung cancer cell lines. Additionally, we identified the T lymphocyte secretory cytokine, IL9, as responsible for the Th9 induced EMT and migratory phenotype of lung cancer cells. This study reveals that specific T lymphocyte subpopulations, i.e. Th9 and Th17 induce EMT in lung cancer cells, suggesting T cell regulated mechanisms of metastasis. Citation Format: Ylia Salazar, Magdalena Huber, Anja Schmall, Werner Seeger, SoniSavai Pullamsetti, Rajkumar Savai, Magdalena Huber, Anja Schmall, Werner Seeger, SoniSavai Pullamsetti. Contribution of Stromal Lymphocytes to Lung Cancer Metastasis: Role in Epithelial Mesenchymal Transition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1594.

Lung cancer is the leading cause of cancer death worldwide. Recent data suggest that cyclic nucleotide phosphodiesterases (PDEs) are relevant in various cancer pathologies. Pathophysiological role of phosphodiesterase 4 (PDE4) with possible therapeutic prospects in lung cancer was investigated. We exposed 10 different lung cancer cell lines (adenocarcinoma, squamous and large cell carcinoma) to hypoxia and assessed expression and activity of PDE4 by real-time PCR, immunocytochemistry, western blotting and PDE activity assays. Expression and activity of distinct PDE4 isoforms (PDE4A and PDE4D) increased in response to hypoxia in eight of the studied cell lines. Furthermore, we analyzed various in silico predicted hypoxia-responsive elements (p-HREs) found in in PDE4A and PDE4D genes. Performing mutation analysis of the p-HRE in luciferase reporter constructs, we identified four functional HRE sites in the PDE4A gene and two functional HRE sites in the PDE4D gene that mediated hypoxic induction of the reporter. Silencing of hypoxia-inducible factor subunits (HIF1α and HIF2α) by small interfering RNA reduced hypoxic induction of PDE4A and PDE4D. Vice versa, using a PDE4 inhibitor (PDE4i) as a cyclic adenosine monophosphate (cAMP) -elevating agent, cAMP analogs or protein kinase A (PKA)-modulating drugs and an exchange protein directly activated by cAMP (EPAC) activator, we demonstrated that PDE4-cAMP-PKA/EPAC axis enhanced HIF signaling as measured by HRE reporter gene assay, HIF and HIF target genes expression ((lactate dehydrogenase A), LDHA, (pyruvate dehydrogenase kinase 1) PDK1 and (vascular endothelial growth factor A) VEGFA). Notably, inhibition of PDE4 by PDE4i or silencing of PDE4A and PDE4D reduced human lung tumor cell proliferation and colony formation. On the other hand, overexpression of PDE4A or PDE4D increased human lung cancer proliferation. Moreover, PDE4i treatment reduced hypoxia-induced VEGF secretion in human cells. In vivo, PDE4i inhibited tumor xenograft growth in nude mice by attenuating proliferation and angiogenesis. Our findings suggest that PDE4 is expressed in lung cancer, crosstalks with HIF signaling and promotes lung cancer progression. Thus, PDE4 may represent a therapeutic target for lung cancer therapy.

Background: Circulating fibrocytes (CFs) are bone marrow derived, mesenchymal progenitor cells that have emerging role in many diseases. CFs was shown to participate in tissue remodeling in pulmonary hypertension and fibrosis, via secretion of different cytokines and growth factors. Nevertheless, their role in the lung cancer still has to be delineated. Thus, our aim is to identify the role of CFs in lung cancer progression and metastatic potential. Results/methods: We generated CFs by isolating human peripheral blood mononuclear cells (PBMCs) and culturing them for 10 days until they differentiated into CFs. Purity (≥95%) of the CFs population was analyzed by flow cytometry and immunofluorescence. Co-culturing of A549 (human alveolar adenocarcinoma cells) and CFs for 12 and 24 hrs, followed by collection of conditioned medium (CM) and RNA from both cells yielded interesting results. Co-cultured CM promoted proliferation (1.31±0.07 versus 1.5±0.13) and migrating capacity (64.6±26.5 versus 472.8±103.4) of A549 tumor cells. In addition, co-cultured CM caused epithelial-to-mesenchymal transition (EMT) of A549 cells; epithelial markers (E-cadherin, cytokeratin) were downregulated, and mesenchymal markers (α-smooth muscle actin, fibronectin) were upregulated. In vivo to study the CFs involvement in lung cancer, we co-injected CFs with A549 cells or A549 cells alone and measured the tumor growth after 28 days. The tumor size was significantly increased (1354.8±333.6 versus 3042.4±373.4) in co-injected group (CF+A549) compared to A549 alone. Screening for genes regulated in co-injected group tumors showed regulation of tumor microenvironment, an increase in macrophage markers (CSF1R, CD68, CD200, MMD), angiogenesis markers (EDNRB, THBS1) and ECM remodeling markers (MMP14, CTSB, CTSH) compared to A549 tumor alone. Conclusions: We believe that circulating fibrocytes may play positive role in the tumor growth and progression. The increase in EMT and migration, may suggest their involvement in invasion and metastasis. Targeting CFs and their secretory molecules can be of therapeutic importance in lung cancer. Citation Format: Alina Asafova, Vandana Nikam, Anja Schmall, Werner Seeger, Robert Voswinckel, Rajkumar Savai. Involvement of circulating fibrocytes in the progression of adenocarcinomas by modulating EMT and tumor microenvironment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2604. doi:10.1158/1538-7445.AM2013-2604

Lung Cancer is the leading cause of cancer deaths worldwide. It is increasingly appreciated that the tumor stroma, consistent of a heterogeneous population of non-neoplastic host cells, is an essential part of cancer initiation, growth and progression. Tumor-associated macrophages (TAMs) can influence cancer progression and metastasis, but the responsible mechanisms remain unclear. Here, we focus on the involvement of bone-marrow derived macrophages (BM- MM) and two of their major chemotactic pathways (CX3CR1-CX3CL1 and CCR2-CCL2 axis) in cancer proliferation and migration. We demonstrate that conditioned medium (CM) derived from co-cultures of lewis lung carcinoma (LLC1) cells with BM- MM (purity >99%) enhances proliferation of LLC1-cells significantly from 1.79±0.1717 to 2.43±0.2067 in an absorbance based BrdU-Assay and a higher colony-number was found in a colony-formation-assay compared to control. In addition LLC1-migration in presence of CM was 5 fold increased compared to controls (172.9±2.18 versus 33.93±2.29). Further, analysis of the cytokine profile of co-culture-derived CM showed regulation of pro-inflammatory cytokines (Interleukin-6 and Interleukin-1α), anti-inflammatory cytokines (Interleukin-10 and Interleukin-1RA) and several chemokines (CCL5 and CCL2). Importantly, evaluation of tumor growth in chemokine receptor knockout mice, CX3CR1-KO and CCR2-KO demonstrated reduced tumor size compared to wild type mice. CX3CR1-KO and CCR2-KO tumor showed reduced in vivo proliferation as assessed by PCNA immunostaining (12.46%±3.188 versus 46.45%±2.922). Further analysis of the tumor microenvironment in CX3CR1-KO and CCR2-KO demonstrated a significant decrease in monocyte/ macrophage accumulation compared to control tumors as assessed by FACS. Compared to the percentage of macrophages in control tumor (7.518±0.78%), a significant decrease was observed in CCR2-KO (2.96± 0.233%) and CX3CR1-KO (3.975±0.6019%) tumors. Based on these findings, we conclude that macrophage plays a crucial role in lung cancer progression and the knowledge of tumor-host interactions can provide novel therapeutic approaches for lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1484. doi:1538-7445.AM2012-1484

Lung cancer is the leading cause of cancer death worldwide. Recent data suggest that cyclic nucleotide phosphodiesterases (PDEs) are relevant in various cancer pathologies. Here, we elucidate the pathophysiological role of PDE4 and its therapeutic prospects in lung cancer. We exposed 10 different non-small cell lung cancer cell lines (adenocarcinoma, squamous, and large cell carcinoma) to hypoxia and assessed the expression and activity of PDE4 by quantitative real-time PCR, immunocytochemistry, western blotting, and PDE activity assays. Expression and activity of distinct PDE4 isoforms (PDE4A and PDE4D) increased in response to hypoxia in eight of the studied cell lines. Furthermore, we determined various potential hypoxia-responsive elements (HRE) in PDE4A and PDE4D. Silencing of hypoxia-inducible factor subunits (HIF1A and HIF2) by siRNA reduced hypoxic induction of PDE4A and PDE4D. Vice versa, using a PDE4 inhibitor (PDE4i), a cAMP-elevating agent, cAMP analogs, PKA activator/inhibitor, and EPAC activator, we demonstrated that PDE4-cAMP-PKA/EPAC axis regulates HIF signaling as measured by HRE reporter gene assay and expression of HIF target genes (LDHA, PDK1, VEGFA). Notably, PDE4i or PDE4A-/PDE4D-selective siRNA reduced human lung tumor cell proliferation in vitro and colony formation and altered the expression of cell-cycle regulators. Moreover, PDE4i treatment reduced hypoxia-induced VEGF secretion in human cells. In vivo, PDE4i inhibited tumor xenograft growth in athymic nude mice by altering proliferation and angiogenesis. Collectively, our findings suggest that PDE4 is overexpressed in lung cancer, exerts crosstalk with HIF pathways, and promotes lung cancer progression; thus, PDE4 may represent a therapeutic target for lung cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1839. doi:1538-7445.AM2012-1839

Lung cancer is frequently complicated by pulmonary infections which may impair prognosis of this disease. Therefore, we investigated the effect of bacterial lipopolysaccharides (LPS) on tumor proliferation in vitro in the non-small cell lung cancer (NSCLC) cell line A549, ex vivo in a tissue culture model using human NSCLC specimens and in vivo in the A549 adenocarcinoma mouse model. LPS induced a time- and dose-dependent increase in proliferation of A549 cells as quantified by MTS activity and cell counting. In parallel, an increased expression of the proliferation marker Ki-67 and cyclooxygenase (COX)-2 was detected both in A549 cells and in ex vivo human NSCLC tissue. Large amounts of COX-2-derived prostaglandin (PG)E(2) were secreted from LPS-stimulated A549 cells. Pharmacological interventions revealed that the proliferative effect of LPS was dependent on CD14 and Toll-like receptor (TLR)4. Moreover, blocking of the epidermal growth factor receptor (EGFR) also decreased LPS-induced proliferation of A549 cells. Inhibition of COX-2 activity in A549 cells severely attenuated both PGE(2) release and proliferation in response to LPS. Synthesis of PGE(2) was also reduced by inhibiting CD14, TLR4 and EGFR in A549 cells. The proliferative effect of LPS on A549 cells could be reproduced in the A549 adenocarcinoma mouse model with enhancement of tumor growth and Ki-67 expression in implanted tumors. In summary, LPS induces proliferation of NSCLC cells in vitro, ex vivo in human NSCLC specimen and in vivo in a mouse model of NSCLC. Pulmonary infection may thus directly induce tumor progression in NSCLC.