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Reduced production of cytokines in PC3 cells subjected to PKCalpha RNAi depletion. IL-8 and GRO were measured in conditioned medium of PC3 cells by ELISA.

Aberrant expression of protein kinase C (PKC) isozymes is a hallmark of cancer. The different members of the PKC family control cellular events associated with cancer development and progression. Whereas the classical/conventional PKCα isozyme has been linked to tumor suppression in most cancer types, here we demonstrate that this kinase is required for the mitogenic activity of aggressive human prostate cancer cells displaying aberrantly high PKCα expression. IHC analysis showed abnormal upregulation of PKCα in human primary prostate tumors. Interestingly, silencing PKCα expression from aggressive prostate cancer cells impairs cell-cycle progression, proliferation, and invasion, as well as their tumorigenic activity in a mouse xenograft model. Mechanistic analysis revealed that PKCα exerts a profound control of gene expression, particularly over genes and transcriptional networks associated with cell-cycle progression and E2F transcription factors. PKCα RNAi depletion from PC3 prostate cancer cells led to a reduction in the expression of proinflammatory cytokine and epithelial-to-mesenchymal transition (EMT) genes, as well as a prominent downregulation of the immune checkpoint ligand PD-L1. This PKCα-dependent gene expression profile was corroborated in silico using human prostate cancer databases. Our studies established PKCα as a multifunctional kinase that plays pleiotropic roles in prostate cancer, particularly by controlling genetic networks associated with tumor growth and progression. The identification of PKCα as a protumorigenic kinase in human prostate cancer provides strong rationale for the development of therapeutic approaches toward targeting PKCα or its effectors.Significance:PKCα was found to be aberrantly expressed in human prostate cancer. Silencing the expression of this kinase from aggressive prostate cancer cell lines reduces their proliferative, tumorigenic, and invasive properties. In addition, our findings implicate PKCα as a major node for transcriptional regulation of tumorigenic, inflammatory, and EMT networks in prostate cancer, highlighting its potential relevance as a therapeutic target.

Pathway and GO enrichment analysis.

Gene Set Enrichment analysis (GSEA) of transcription factor binding sites (TFBS).

Relationship between PRKCA and AR expression in prostate cancer. Correlation graphs for individual datasets as well as a meta-analysis are shown.

Differentially expressed genes in PKCalpha silenced PC3 cells.

Time-dependent expression of PKCalpha in prostate cancer cells after RNAi silencing with three different siRNA duplexes.

Effect of PKCalpha depletion using shRNA lentiviruses on PC3 cell invasion, as determined with a Boyden chamber.

Effect of treatment with Cdk4/6 and Cdk4 inhibitors in combination with PKCα RNAi silencing on prostate cancer cell viability.

Cell cycle analysis upon PKCalpha RNAi silencing. Representative Rb phosphorylation as well as cell cycle distribution analysis by FACS in PKCalpha silenced PC3 cells are shown.

PDF file - 76K, Summary statistics for Fig. 1C and Fig.2A

PDF file - 410K, Calcium influx in myeloid cells induced by CCL1 and SDF1

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PDF file - 75K, Summary statistics for Figure 1

PDF file - 70K, CCR8+ myeloid cells induce FoxP3 expression in T cells

Purpose:Fluorescence-guided surgery using tumor-targeted contrast agents has been developed to improve the completeness of oncologic resections. Quenched activity–based probes that fluoresce after covalently binding to tumor-specific enzymes have been proposed to improve specificity, but none have been tested in humans. Here, we report the successful clinical translation of a cathepsin activity–based probe (VGT-309) for fluorescence-guided surgery.Experimental Design:We optimized the specificity, dosing, and timing of VGT-309 in preclinical models of lung cancer. To evaluate clinical feasibility, we conducted a canine study of VGT-309 during pulmonary tumor resection. We then conducted a randomized, double-blind, dose-escalation study in healthy human volunteers receiving VGT-309 to evaluate safety. Finally, we tested VGT-309 in humans undergoing lung cancer surgery.Results:In preclinical models, we found highly specific tumor cell labeling that was blocked by a broad spectrum cathepsin inhibitor. When evaluating VGT-309 for guidance during resection of canine tumors, we found that the probe selectively labeled tumors and demonstrated high tumor-to-background ratio (TBR; range: 2.15–3.71). In the Phase I human study, we found that VGT-309 was safe at all doses studied. In the ongoing Phase II trial, we report two cases in which VGT-309 localized visually occult, non-palpable tumors (TBRs = 2.83 and 7.18) in real time to illustrate its successful clinical translation and potential to improve surgical management.Conclusions:This first-in-human study demonstrates the safety and feasibility of VGT-309 to label human pulmonary tumors during resection. These results may be generalizable to other cancers due to cathepsin overexpression in many solid tumors.

Supplementary Data from A Cathepsin-Targeted Quenched Activity–Based Probe Facilitates Enhanced Detection of Human Tumors during Resection

Expression of inhibitory receptors on tumor vs. spleen infiltrating lymphocytes.

Purpose: Tumor-infiltrating lymphocytes (TILs) become hypofunctional, although the mechanisms are not clear. Our goal was to generate a model of human tumor-induced TIL hypofunction to study mechanisms and to test anti-human therapeutics.Experimental Design: We transduced human T cells with a published, optimized T-cell receptor (TCR) that is directed to a peptide within the cancer testis antigen, NY-ESO-1. After demonstrating antigen-specific in vitro activity, these cells were used to target a human lung cancer line that expressed NY-ESO-1 in the appropriate HLA context growing in immunodeficient mice. The ability of anti-PD1 antibody to augment efficacy was tested.Results: Injection of transgenic T cells had some antitumor activity, but did not eliminate the tumors. The injected T cells became profoundly hypofunctional accompanied by upregulation of PD1, Tim3, and Lag3 with coexpression of multiple inhibitory receptors in a high percentage of cells. This model allowed us to test reagents targeted specifically to human T cells. We found that injections of an anti-PD1 antibody in combination with T cells led to decreased TIL hypofunction and augmented the efficacy of the adoptively transferred T cells.Conclusions: This model offers a platform for preclinical testing of adjuvant immunotherapeutics targeted to human T cells prior to transition to the bedside. Because the model employs engineering of human T cells with a TCR clone instead of a CAR, it allows for study of the biology of tumor-reactive TILs that signal through an endogenous TCR. The lessons learned from TCR-engineered TILs can thus be applied to tumor-reactive TILs. Clin Cancer Res; 22(2); 436–47. ©2015 AACR.See related commentary by Yang, p. 275

Description of cell culture conditions, isolation/activation/transduction/expansion of T cells, generation of target cell lines, flow cytometric conditions, ELISA measurements, statistical analyses, and animal use.

Co-expression of inhibitory receptors on tumor infiltrating lymphocytes.

PDF file - 57K, Summary statistics for Fig.4A

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Comparison of inhibitory receptors on CD8 and CD4 tumor infiltrating lymphocytes.

Summary of publications describing tumor induced hypofunction of human tumor infiltrating lymphocytes.

PDF file - 51K, CCL1-induced IL6 production

Expression of PDL1 on tumors.

PDF file - 169K, CCR8 expression in myeloid cells isolated from blood of cancer patients

Purpose: Chemokines are involved in cancer-related inflammation and malignant progression. In this study, we evaluated expression of CCR8 and its natural cognate ligand CCL1 in patients with urothelial carcinomas of bladder and renal cell carcinomas.Experimental Design: We examined CCR8 expression in peripheral blood and tumor tissues from patients with bladder and renal carcinomas. CCR8-positive myeloid cells were isolated from cancer tissues with magnetic beads and tested in vitro for cytokine production and ability to modulate T-cell function.Results: We show that monocytic and granulocytic myeloid cell subsets in peripheral blood of patients with cancer with urothelial and renal carcinomas display increased expression of chemokine receptor CCR8. Upregulated expression of CCR8 is also detected within human cancer tissues and primarily limited to tumor-associated macrophages. When isolated, CD11b+CCR8+ cell subset produces the highest levels of proinflammatory and proangiogenic factors among intratumoral CD11b myeloid cells. Tumor-infiltrating CD11b+CCR8+ cells selectively display activated Stat3 and are capable of inducing FoxP3 expression in autologous T lymphocytes. Primary human tumors produce substantial amounts of the natural CCR8 ligand CCL1.Conclusions: This study provides the first evidence that CCR8+ myeloid cell subset is expanded in patients with cancer. Elevated secretion of CCL1 by tumors and increased presence of CCR8+ myeloid cells in peripheral blood and cancer tissues indicate that CCL1/CCR8 axis is a component of cancer-related inflammation and may contribute to immune evasion. Obtained results also implicate that blockade of CCR8 signals may provide an attractive strategy for therapeutic intervention in human urothelial and renal cancers. Clin Cancer Res; 19(7); 1670–80. ©2013 AACR.

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Supplementary materials and methods

Supplementary Figures 1-8. Supplementary Fig. 1. CD11b+Gr1+ populations were isolated by FACS from spleens of tumor-bearing p120-/- and control mice. Supplementary Fig. 2. CD38 is expressed by the CD11b+Gr1+ population in a murine model of Barrett's Esophagus. Supplementary Fig. 3. The HNM007 tumors induce CD38 expression on MDSC population similarly to the autochthonous p120-/- tumors. Supplementary Fig. 4. Immunohistochemical analysis of HNM007 tumors co-injected with CD38High or CD38Low MDSCs. Supplementary Fig. 5. CD38high CD11b+Gr1+ cells produce higher levels of iNOS and pNFκB. Supplementary Fig.6. Network analysis of differentially regulated genes in CD38high and CD38low MDSCs shows upregulation of RB1 pathway. Supplementary Fig.7. Distribution of G-MDSC, M-MDSC and mature monocytes is perturbed in tumor-bearing Cd38-/- mice. Supplementary Fig. 8. CD38 is expressed on human MDSC-like cell population that is expanded in the peripheral blood of advanced-stage cancer patients.

Supplementary Tables 1 and 2. Supplementary Table 1. List of the genes differentially expressed in CD11b+Gr1+ splenocytes from tumor-bearing, compared to control mice. Supplementary Table 2. List of the genes differentially expressed in CD38high, compared to CD38low, CD11b+Gr1+ splenocytes from tumor-bearing mice.

Aberrant expression of protein kinase C (PKC) isozymes is a hallmark of cancer. The different members of the PKC family control cellular events associated with cancer development and progression. Whereas the classical/conventional PKCα isozyme has been linked to tumor suppression in most cancer types, here we demonstrate that this kinase is required for the mitogenic activity of aggressive human prostate cancer cells displaying aberrantly high PKCα expression. IHC analysis showed abnormal upregulation of PKCα in human primary prostate tumors. Interestingly, silencing PKCα expression from aggressive prostate cancer cells impairs cell-cycle progression, proliferation, and invasion, as well as their tumorigenic activity in a mouse xenograft model. Mechanistic analysis revealed that PKCα exerts a profound control of gene expression, particularly over genes and transcriptional networks associated with cell-cycle progression and E2F transcription factors. PKCα RNAi depletion from PC3 prostate cancer cells led to a reduction in the expression of proinflammatory cytokine and epithelial-to-mesenchymal transition (EMT) genes, as well as a prominent downregulation of the immune checkpoint ligand PD-L1. This PKCα-dependent gene expression profile was corroborated in silico using human prostate cancer databases. Our studies established PKCα as a multifunctional kinase that plays pleiotropic roles in prostate cancer, particularly by controlling genetic networks associated with tumor growth and progression. The identification of PKCα as a protumorigenic kinase in human prostate cancer provides strong rationale for the development of therapeutic approaches toward targeting PKCα or its effectors. Significance: PKCα was found to be aberrantly expressed in human prostate cancer. Silencing the expression of this kinase from aggressive prostate cancer cell lines reduces their proliferative, tumorigenic, and invasive properties. In addition, our findings implicate PKCα as a major node for transcriptional regulation of tumorigenic, inflammatory, and EMT networks in prostate cancer, highlighting its potential relevance as a therapeutic target.

PPM1D/Wip1 is a negative regulator of the tumor suppressor p53 and is overexpressed in several human solid tumors. Recent reports associate gain-of-function mutations of PPM1D in immune cells with worse outcomes for several human cancers. Here we show that mice with genetic knockout of Ppm1d or with conditional knockout of Ppm1d in the hematopoietic system, in myeloid cells, or in neutrophils all display significantly reduced growth of syngeneic melanoma or lung carcinoma tumors. Ppm1d knockout neutrophils infiltrate tumors extensively. Chemical inhibition of Wip1 in human or mouse neutrophils increases anti-tumor phenotypes, p53-dependent expression of co-stimulatory ligands, and proliferation of co-cultured cytotoxic T cells. These results suggest that inhibition of Wip1 in neutrophils enhances immune anti-tumor responses., Wip1 is a negative regulator of the tumor suppressor p53 and is overexpressed in several human cancers. Here the authors show that inactivation of Wip1 in neutrophils promotes p53-dependent expression of co-stimulatory ligands and anti-tumor immune responses, reducing tumor growth in preclinical cancer models.

Transforming growth factor-β (TGFβ) signaling is common in many solid tumors and is initiated by binding of the high affinity canonical ligands TGFβ1, 2, οr 3 to TGFβR2, which forms a heteromeric receptor complex with TGFβR1 (Derynck et al, Nature Review Clinical Oncology 2020). Activation of the pathway results in potent suppression of immune cell-mediated anti-tumor immunity and has been reported to predict poor response to PD-(L)1 targeted therapy in patients (Mariathasan et al, Nature 2018; Kieffer et al, Cancer Discovery 2020). However, TGFβ drug development has been hampered by the occurrence of adverse events. INCA33890 is a dual PD-1 and TGFβR2 binding bispecific Biclonics® antibody, developed to antagonize the TGFβ signaling pathway specifically in cells co-expressing PD-1 and TGFBR2. Additionally, it potently antagonizes the PD-1 axis independently of TGFβR2 co-expression. The cell-selective action of INCA33890 was designed to mitigate risks of the known adverse effects associated with TGFβ-pathway inhibition in tissues requiring active TGFβ signaling. ΙΝCΑ33890 mediates its specificity through a PD-1 binding arm with a >10-fold higher affinity relative to the TGFβR2 binding arm. Consistent with this profile, in isogenic Jurkat cells expressing TGFβR2 ± PD-1, INCA33890 potently inhibits TGFβ1-induced pSMAD activation in a PD-1-correlated manner. Additionally, in two independent PD-1 reporter assays, INCA33890 inhibited SHP recruitment and enhanced NFAT activation with a potency within an order of magnitude to that of pembrolizumab. In mixed lymphocyte reaction assays with exhausted primary human T-cells, INCA33890 was found to induce a similar level of anti-tumor cytokine production as the combination of pembrolizumab and an anti-TGFβR2 antagonist mAb. Treatment of primary ovarian ascites with INCA33890 ex vivo induced IFNγ production in all donors tested, while pembrolizumab had no activity. Similarly, in human CD34+ cell-engrafted NSG mice, INCA33890 significantly inhibited the growth of human MDA-MB-231 and A375 subcutaneous xenograft tumors, whereas pembrolizumab or an anti-TGFβR2 antibody had little or no monotherapy activity. INCA33890 had a balanced pharmacokinetic and potency profile and was well tolerated in NHPs at exposures required for pharmacodynamic and tumor growth inhibiting activity in rodents. Encouragingly, there was no evidence of adverse effects in NHPs due to TGFβ-pathway blockade. Collectively, these results provide compelling data for an effective and specific approach to simultaneously antagonizing TGFβ and PD-1 signaling in tumors. Clinical development of INCA33890 in checkpoint inhibitor-resistant and other cancers has been initiated. Citation Format: Liang-Chuan S. Wang, Rinse Klooster, Ashwini Kulkarni, Amaya Garcia de Vinuesa, Maxim Soloviev, Linda JA Hendriks, Lu Huo, Michael Weber, Arpita Mondal, Yonghong Zhao, Shane Harvey, Xin He, Hong Chang, April Horsey, Alla Volgina, Yue Zhang, Veethika Pandey, Yan-Ou Yang, Jonathan Rios-Doria, Evgeniy Eruslanov, Daniel J. Powell, Steven M. Albelda, John de Kruif, Horacio Nastri, Cecile Geuijen, Patrick A. Mayes. INCA33890, a novel PD-1×TGFꞵR2 bispecific antibody conditionally antagonizes TGFꞵ signaling in primary immune cells co-expressing PD-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2936.

Neutrophils are the first responders to infection and inflammation and are thus a critical component of innate immune defense. Understanding the behavior of neutrophils as they act within various inflammatory contexts has provided insights into their role in sterile and infectious diseases; however, the field of neutrophils in cancer is comparatively young. Here, we summarize key concepts and current knowledge gaps related to the diverse roles of neutrophils throughout cancer progression. We discuss sources of neutrophil heterogeneity in cancer and provide recommendations on nomenclature for neutrophil states that are distinct in maturation and activation. We address discrepancies in the literature that highlight a need for technical standards that ought to be considered between laboratories. Finally, we review emerging questions in neutrophil biology and innate immunity in cancer. Overall, we emphasize that neutrophils are a more diverse population than previously appreciated and that their role in cancer may present novel unexplored opportunities to treat cancer. Acknowledgments This consensus statement is dedicated to our esteemed colleagues, Zena Werb and Paul S. Frenette, who attended and participated in the Banbury Center meeting and encouraged a collective effort by attendees to write this report. Both made significant contributions to ideas and discussions that are represented here. The Banbury Center meeting was organized by M. Egeblad, P. Kubes, K.E. de Visser, R. Leshan, and Banbury Center staff. The meeting was supported financially by Cold Spring Harbor Laboratory Northwell Health Affiliation. The funder had no involvement with the writing of this consensus statement. D.F. Quail acknowledges funding from the Canadian Institutes of Health Research (PJT-159742, PJT-178306), Terry Fox Research Institute, and Tier II Canada Research Chair. B. Amulic acknowledges funding from the Medical Research Council (MR/R02149x/1). M. Aziz acknowledges funding from the National Institutes of Health (NIH; R01GM129633). B.J. Barnes acknowledges funding from the NIH National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR065959-01) and Department of Defense (CDMRP LRP W81XWH-18-1-0674). E. Eruslanov acknowledges funding from the NIH National Cancer Institute (NCI; R01CA187392) and the Department of Defense (CDMRP W81XWH-15-1-0717). Z.G. Fridlender acknowledges funding from the the Israel Science Foundation (grant number 1708/20) and the Sasson and Luisa Naor Fund. H.S. Goodridge acknowledges funding from the NIH National Institute of Allergy and Infectious Diseases (R01AI134987). Z. Granot acknowledges funding from the Israel Science Foundation Grant 405/18, the Israel Cancer Research Fund, the Deutsche Forschungsgemeinschaft, and the Rosetrees Trust. A. Hidalgo acknowledges funding from FET-OPEN (861878) from the European Commission; the Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the Ministerio de Ciencia e Innovacion and the Pro-CNIC Foundation. M.J. Kaplan acknowledges funding from the Intramural Research Program of the NIH, National Institute of Arthritis and Musculoskeletal and Skin Diseases (ZIAAR041199). I. Malanchi acknowledges funding from the European Research Council grant (ERC CoG-H2020-725492) and from the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001112), the UK Medical Research Council (FC001112), and the Wellcome Trust (FC001112). T. Merghoub acknowledges funding from the NIH/NCI Cancer Center Support Grant (P30 CA008748/NCI R01 CA056821), Swim Across America, Ludwig Institute for Cancer Research, Ludwig Center for Cancer Immunotherapy at Memorial Sloan Kettering, Cancer Research Institute, and Parker Institute for Cancer Immunotherapy. E. Meylan acknowledges funding from the Swiss National Science Foundation (310030_179324) and Fonds de la Recherche Scientifique (MISU F.6003.22). M.J. Pittet acknowledges funding from the Institut Suisse de Recherche Experimentale sur le Cancer Foundation, Ludwig Cancer Research, and the NIH (P01CA240239, R01CA218579). I.A. Udalova acknowledges funding from the Wellcome Trust Investigator Award (209422/Z/17/Z). T.K. van den Berg acknowledges funding from Byondis BV, the Dutch Ministry of Health, and the Dutch Cancer Society (10300). D.D. Wagner acknowledges funding from the NIH National Health, Lung and Blood Institute (R35HL135765). P. Wang acknowledges funding from the NIH National Institute of General Medical Sciences (R35GM118337). A. Zychlinsky acknowledges funding from the Max Planck Society. K.E. de Visser acknowledges funding from the Dutch Cancer Society (KWF10623, KWF10083, and KWF13191), Oncode Institute, and the Netherlands Organization for Scientific Research (NWO-VICI 91819616). M. Egeblad acknowledges funding from the Department of Defense, Congressional Directed Medical Research Program (W81XWH2010753). P. Kubes acknowledges funding from the Canadian Institutes of Health Research. Sí

Purpose: Fluorescence-guided surgery using tumor-targeted contrast agents has been developed to improve the completeness of oncologic resections. Quenched activity–based probes that fluoresce after covalently binding to tumor-specific enzymes have been proposed to improve specificity, but none have been tested in humans. Here, we report the successful clinical translation of a cathepsin activity–based probe (VGT-309) for fluorescence-guided surgery. Experimental Design: We optimized the specificity, dosing, and timing of VGT-309 in preclinical models of lung cancer. To evaluate clinical feasibility, we conducted a canine study of VGT-309 during pulmonary tumor resection. We then conducted a randomized, double-blind, dose-escalation study in healthy human volunteers receiving VGT-309 to evaluate safety. Finally, we tested VGT-309 in humans undergoing lung cancer surgery. Results: In preclinical models, we found highly specific tumor cell labeling that was blocked by a broad spectrum cathepsin inhibitor. When evaluating VGT-309 for guidance during resection of canine tumors, we found that the probe selectively labeled tumors and demonstrated high tumor-to-background ratio (TBR; range: 2.15–3.71). In the Phase I human study, we found that VGT-309 was safe at all doses studied. In the ongoing Phase II trial, we report two cases in which VGT-309 localized visually occult, non-palpable tumors (TBRs = 2.83 and 7.18) in real time to illustrate its successful clinical translation and potential to improve surgical management. Conclusions: This first-in-human study demonstrates the safety and feasibility of VGT-309 to label human pulmonary tumors during resection. These results may be generalizable to other cancers due to cathepsin overexpression in many solid tumors.

Gene-mediated cytotoxic immunotherapy (GMCI) is an immuno-oncology approach involving local delivery of a replication-deficient adenovirus expressing herpes simplex thymidine kinase (AdV-tk) followed by anti-herpetic prodrug activation that promotes immunogenic tumor cell death, antigen-presenting cell activation, and T cell stimulation. This phase I dose-escalation pilot trial assessed bronchoscopic delivery of AdV-tk in patients with suspected lung cancer who were candidates for surgery. A single intra-tumoral AdV-tk injection in three dose cohorts (maximum 1012 viral particles) was performed during diagnostic staging, followed by a 14-day course of the prodrug valacyclovir, and subsequent surgery 1 week later. Twelve patients participated after appropriate informed consent. Vector-related adverse events were minimal. Immune biomarkers were evaluated in tumor and blood before and after GMCI. Significantly increased infiltration of CD8+ T cells was found in resected tumors. Expression of activation, inhibitory, and proliferation markers, such as human leukocyte antigen (HLA)-DR, CD38, Ki67, PD-1, CD39, and CTLA-4, were significantly increased in both the tumor and peripheral CD8+ T cells. Thus, intratumoral AdV-tk injection into non-small-cell lung cancer (NSCLC) proved safe and feasible, and it effectively induced CD8+ T cell activation. These data provide a foundation for additional clinical trials of GMCI for lung cancer patients with potential benefit if combined with other immune therapies., Graphical Abstract, Predina and colleagues describe a phase I dose-escalation trial using bronchoscopic delivery of a replication-deficient adenovirus expressing herpes simplex thymidine kinase (AdV-tk) in lung cancer patients who underwent surgery. AdV-tk injection proved safe, feasible, and effectively induced CD8+ T cell activation in blood and in resected tumor tissues.

Cancer progression is marked by dysfunctional tumor-infiltrating lymphocytes (TIL) with high inhibitory receptor (IR) expression. Because IR blockade has led to clinical responses in some patients with non–small cell lung cancer (NSCLC), we investigated how IRs influenced CD8+ TIL function from freshly digested early-stage NSCLC tissues using a killing assay and intracellular cytokine staining after in vitro T-cell restimulation. Early-stage lung cancer TIL function was heterogeneous with only about one third of patients showing decrements in cytokine production and lytic function. TIL hypofunction did not correlate with clinical factors, coexisting immune cells (macrophages, neutrophils, or CD4+ T regulatory cells), nor with PD-1, TIGIT, TIM-3, CD39, or CTLA-4 expression. Instead, we found that the presence of the integrin αeβ7 (CD103), characteristic of tissue-resident memory cells (TRM), was positively associated with cytokine production, whereas expression of the transcription factor Eomesodermin (Eomes) was negatively associated with TIL function. These data suggest that the functionality of CD8+ TILs from early-stage NSCLCs may be influenced by competition between an antitumor CD103+ TRM program and an exhaustion program marked by Eomes expression. Understanding the mechanisms of T-cell function in the progression of lung cancer may have clinical implications for immunotherapy.

Soft-tissue sarcomas (STS) are a group of lethal mesenchymal tumors that are a major cause of cancer related morbidity for those under the age of 20.PI3K inhibitors have been found to be effective against subset of STS but are associated with significant toxicities limiting their use.We have thought to test a selective fibroblast activation protein (FAP) targeted delivery of PI3K/AKT/mTOR inhibitor omipalisib to STS in pre-clinical setting as FAP is overwhelmingly expressed agressive soft tissue malignancies. Experimental Design: Patient derived fibrosarcomas (aSMA-,FAP+,pAKT+) were isolated by flow cytometry.FAP(-)HT1080 fibrosarcoma cell lines were used as a negative control whereas methylcoanthrene induced mouse fibrosarcoma transfected with human FAP cDNA were used as positive control.Cell expression of FAP,AKT,mTOR was compared using SDS Page,IHC,and flow cytometry.FAP ligand coupled to omipalisib(FAPL-PI3Ki)was then evaluated in-vitro and in small animal models. Results: Cell lines with known FAP expression demonstrated significant uptake of FAPL conjugated NIR fluorochrome.Wild type cells(hFAP-) showed no ligand binding both in flow cytometry and fluorescent microscopy(p Citation Format: Feredun Azari, Gregory T. Kennedy, Ashley Chang, Bilal Nadeem, Azra Din, Isvita Marfatia, Steven Albelda, Philip Low, Madduri Srinivasarao, Ramesh Mukkamala, Neil T. Sullivan, Evgeniy Eruslanov, Sunil Singhal. Selective delivery of fibroblast activation protein conjugated dual phosphoinositide 3-kinase–AKT Kinase-mTOR inhibitor associated with decreased tumor proliferation and on-target toxicity in high-grade soft-tissue sarcomas [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A016.

Veglia et al. characterize the heterogeneity of polymorphonuclear neutrophils (PMNs) in the tumor microenvironment, spleen, and peripheral blood. The identification of neutrophil populations with potent immune suppressive activity opens selective targeting opportunities., In this study, using single-cell RNA-seq, cell mass spectrometry, flow cytometry, and functional analysis, we characterized the heterogeneity of polymorphonuclear neutrophils (PMNs) in cancer. We describe three populations of PMNs in tumor-bearing mice: classical PMNs, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), and activated PMN-MDSCs with potent immune suppressive activity. In spleens of mice, PMN-MDSCs gradually replaced PMNs during tumor progression. Activated PMN-MDSCs were found only in tumors, where they were present at the very early stages of the disease. These populations of PMNs in mice could be separated based on the expression of CD14. In peripheral blood of cancer patients, we identified two distinct populations of PMNs with characteristics of classical PMNs and PMN-MDSCs. The gene signature of tumor PMN-MDSCs was similar to that in mouse activated PMN-MDSCs and was closely associated with negative clinical outcome in cancer patients. Thus, we provide evidence that PMN-MDSCs are a distinct population of PMNs with unique features and potential for selective targeting opportunities.

Objective: Cancer-associated fibroblasts (CAFs) are a key component of the tumor microenvironment and affect neoplastic progression by stromal remodeling, paracrine signaling, potentiating cancer cell proliferation, and immune system evasion. As such, they are a potential target for optimizing therapeutic and diagnostic strategies. The purpose of our study was to evaluate if Fibroblast Activation Protein (FAP) can be a suitable target for molecular imaging guided cancer localization during surgery because FAP is overwhelmingly expressed by CAFs. Methods: Patient derived lung adenocarcinoma FAP+/alpha SMA+ myofibroblasts (CAFs) were isolated by flow cytometry and cultured according to institutional best practices. Normal fibroblasts from 3T3 cell line were used as negative control whereas normal lung fibroblasts transfected with human FAP cDNA were used as positive control. Cell expression of FAP was compared using SDS Page analysis and flow cytometry. FAP ligand coupled to NIR S0456 fluorochrome (FAP-S0456) was then evaluated with in-vitro dose escalation, cell-toxicity study, fluorescence parameters, and immunofluorescence microscopy. Results: Patient derived lung cancer (LC) CAFs had lower FAP-S0456 dye binding expression compared to transfected fibroblasts (TF), while normal fibroblasts (FAP-, aSMA-) had no FAP-S0456 dye binding detected on FACS direct antibody analysis (MFI 1033 vs 244, p Conclusion: FAP transmembrane receptor is highly expressed in cancer associated fibroblasts and can serve as a potential target for FAP ligand conjugated near-infrared tracers. This study demonstrates proof of principle of utilizing FAP-S0456 in CAF in-vitro models and paves a path for pre-clinical diagnostic and therapeutic validation for surgical applications. Citation Format: Feredun Azari, Gregory Kennedy, Ashley Chang, Bilal Nadeem, Neil Sullivan, Emanuel Encarnado, Steven Albelda, Evgeniy Eruslanov, Sunil Singhal. Fibroblast activation protein transmembrane receptor targeted near-infrared fluorochrome specifically identifies patient derived lung cancer associated fibroblasts in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4142.

Giant cell arteritis (GCA) is a common form of primary systemic vasculitis in adults, with no reliable indicators of prognosis or treatment responses. We used single cell technologies to comprehensively map immune cell populations in the blood of patients with GCA and identified the CD66b+CD15+CD10lo/–CD64– band neutrophils and CD66bhiCD15+CD10lo/–CD64+/bright myelocytes/metamyelocytes to be unequivocally associated with both the clinical phenotype and response to treatment. Immature neutrophils were resistant to apoptosis, remained in the vasculature for a prolonged period of time, interacted with platelets, and extravasated into the tissue surrounding the temporal arteries of patients with GCA. We discovered that immature neutrophils generated high levels of extracellular reactive oxygen species, leading to enhanced protein oxidation and permeability of endothelial barrier in an in vitro coculture system. The same populations were also detected in other systemic vasculitides. These findings link functions of immature neutrophils to disease pathogenesis, establishing a clinical cellular signature of GCA and suggesting different therapeutic approaches in systemic vascular inflammation., Bona fide immature neutrophil subsets produce unchecked extracellular ROS that contributes to vascular pathologies.