Darren R. Cullinan

Tissue-Resident Macrophages in Pancreatic Ductal Adenocarcinoma Originate from Embryonic Hematopoiesis and Promote Tumor Progression

&NA; Tumor‐associated macrophages (TAMs) are essential components of the cancer microenvironment and play critical roles in the regulation of tumor progression. Optimal therapeutic intervention requires in‐depth understanding of the sources that sustain macrophages in malignant tissues. In this study, we investigated the ontogeny of TAMs in murine pancreatic ductal adenocarcinoma (PDAC) models. We identified both inflammatory monocytes and tissue‐resident macrophages as sources of TAMs. Unexpectedly, significant portions of pancreas‐resident macrophages originated from embryonic development and expanded through in situ proliferation during tumor progression. Whereas monocyte‐derived TAMs played more potent roles in antigen presentation, embryonically derived TAMs exhibited a pro‐fibrotic transcriptional profile, indicative of their role in producing and remodeling molecules in the extracellular matrix. Collectively, these findings uncover the heterogeneity of TAM origin and functions and could provide therapeutic insight for PDAC treatment. Graphical Abstract Figure. No caption available. HighlightsTAMs in PDAC are derived from both monocytes and embryonic macrophagesTissue‐resident embryonic macrophages promote PDAC progressionEmbryonically derived tissue‐resident TAMs expand in PDAC via in situ proliferationEmbryonically derived TAMs exhibit unique pro‐fibrotic activities &NA; Zhu et al. identify tissue‐resident macrophages of embryonic origin as a source of tumor‐associated macrophages in pancreatic ductal adenocarcinoma. These cells expand through in situ proliferation during tumor progression and demonstrate a unique pro‐fibrotic transcriptional profile distinct from that of their monocyte‐derived counterparts.

Targeting both tumour-associated CXCR2+ neutrophils and CCR2+ macrophages disrupts myeloid recruitment and improves chemotherapeutic responses in pancreatic ductal adenocarcinoma

Objective Chemokine pathways are co-opted by pancreatic adenocarcinoma (PDAC) to facilitate myeloid cell recruitment from the bone marrow to establish an immunosuppressive tumour microenvironment (TME). Targeting tumour-associated CXCR2+neutrophils (TAN) or tumour-associated CCR2+ macrophages (TAM) alone improves antitumour immunity in preclinical models. However, a compensatory influx of an alternative myeloid subset may result in a persistent immunosuppressive TME and promote therapeutic resistance. Here, we show CCR2 and CXCR2 combined blockade reduces total tumour-infiltrating myeloids, promoting a more robust antitumour immune response in PDAC compared with either strategy alone. Methods Blood, bone marrow and tumours were analysed from PDAC patients and controls. Treatment response and correlative studies were performed in mice with established orthotopic PDAC tumours treated with a small molecule CCR2 inhibitor (CCR2i) and CXCR2 inhibitor (CXCR2i), alone and in combination with chemotherapy. Results A systemic increase in CXCR2+ TAN correlates with poor prognosis in PDAC, and patients receiving CCR2i showed increased tumour-infiltrating CXCR2+ TAN following treatment. In an orthotopic PDAC model, CXCR2 blockade prevented neutrophil mobilisation from the circulation and augmented chemotherapeutic efficacy. However, depletion of either CXCR2+ TAN or CCR2+ TAM resulted in a compensatory response of the alternative myeloid subset, recapitulating human disease. This was overcome by combined CCR2i and CXCR2i, which augmented antitumour immunity and improved response to FOLFIRINOX chemotherapy. Conclusion Dual targeting of CCR2+ TAM and CXCR2+ TAN improves antitumour immunity and chemotherapeutic response in PDAC compared with either strategy alone.

Conjugation to the sigma-2 ligand SV119 overcomes uptake blockade and converts dm-Erastin into a potent pancreatic cancer therapeutic.

Cancer-selective drug delivery is an important concept in improving treatment while minimizing off-site toxicities, and sigma-2 receptors, which are overexpressed in solid tumors, represent attractive pharmacologic targets. Select sigma-2 ligands have been shown to be rapidly internalized selectively into cancer cells while retaining the capacity to deliver small molecules as drug cargoes. We utilized the sigma-2-based drug delivery concept to convert Erastin, a clinically underperforming drug, into a potent pancreatic cancer therapeutic. The Erastin derivative des-methyl Erastin (dm-Erastin) was chemically linked to sigma-2 ligand SV119 to create SW V-49. Conjugation increased the killing capacity of dm-Erastin by nearly 35-fold in vitro and reduced the size of established tumors and doubled the median survival in syngeneic and patient-derived xenograft models when compared to non-targeted dm-Erastin. Mechanistic analyses demonstrated that cell death was associated with robust reactive oxygen species production and could be efficiently antagonized with antioxidants. Mass spectrometry was employed to demonstrate selective uptake into pancreatic cancer cells. Thus, targeted delivery of dm-Erastin via conjugation to the sigma-2 ligand SV119 produced efficient tumor control and prolonged animal survival with minimal off-target toxicities, and SW V-49 represents a promising new therapeutic with the potential to advance the fight against pancreatic cancer.

Fundamentals of Laparoscopic Surgery: Not Only for Senior Residents

OBJECTIVE Fundamentals of laparoscopic surgery (FLS) was developed by the Society of American Gastrointestinal and Endoscopic Surgeons to teach the physiology, fundamental knowledge, and technical skills required for basic laparoscopic surgery. We hypothesize that residents are doing more laparoscopic surgery earlier in residency, and therefore would benefit from an earlier assessment of basic laparoscopic skills. Here, we examine FLS test results and ACGME case logs to determine whether it is practical to administer FLS earlier in residency. DESIGN FLS test results were reviewed for the 42 residents completing FLS between July 2011 and July 2016. ACGME case logs for current and former residents were reviewed for laparoscopic cases logged by each postgraduate year. Basic and complex laparoscopic cases were determined by ACGME General Surgery Defined Category and Minimums Report. Descriptive statistics were used for analysis. SETTING Academic general surgery residency, Washington University in St. Louis School of Medicine. PARTICIPANTS Current and former general surgery residents. RESULTS A total of 42 residents took and passed FLS between July 2011 and July 2016. All residents successfully passed the FLS knowledge and skills examinations on the first attempt regardless of their postgraduate year (PGY 3n = 13, PGY 4n = 15, and PGY 5n = 14). Total laparoscopic case volume has increased over time. Residents who graduated in 2012 or 2013 completed 229 laparoscopic cases compared to 267 laparoscopic cases for those who graduated from 2014 to 2016 (p = 0.02). Additionally, current residents completed more laparoscopic cases in the first 2 years of residency than residents who graduated from 2012 to 2016 (median current = 38; former = 22; p < 0.001). Examining laparoscopic case numbers for current residents by PGY demonstrated that the number of total and complex laparoscopic cases increased in each of the first 3 years of residency with the largest increase occurring between the PGY 2 and PGY 3 years. In the PGY 4 and PGY 5 years, most laparoscopic cases were complex. CONCLUSION Increased use of laparoscopic surgery has led to a corresponding increase in laparoscopic case volume among general surgery residents. We would advocate for FLS testing to serve as an early assessment of laparoscopic knowledge and skill and should be performed before a significant increase in complex laparoscopic surgery during training.

Interim Analysis of a Prospective Multi-Institutional Study of Surgery Resident Experience with Flexibility in Surgical Training

BACKGROUND The Flexibility in Surgical Training (FIST) consortium project was designed to evaluate the feasibility and resident outcomes of optional subspecialty-focused training within general surgery residency training. STUDY DESIGN After approval by the American Board of Surgery, R4 and R5 residents were permitted to customize up to 12 of the final 24 months of residency for early tracking into 1 of 9 subspecialty tracks. A prospective IRB-approved study was designed across 7 institutions to evaluate the impact of this option on operative experience, in-service exam (American Board of Surgery In-Training Examination [ABSITE]) and ACGME milestone performance, and resident and program director (PD) perceptions. The FIST residents were compared with chief residents before FIST initiation (controls) as well as residents during the study period who did not participate in FIST (no specialization track, NonS). RESULTS From 2013 to 2017, 122 of 214 chief residents (57%) completed a FIST subspecialty track. There were no differences in median ABSITE scores between FIST, NonS residents, and controls. The ACGME milestones at the end of the R5 year favored the FIST residents in 13 of 16 milestones compared with NonS. Case logs demonstrated an increase in track-specific cases compared with NonS residents. Resident and PD surveys reported a generally favorable experience with FIST. CONCLUSIONS In this prospective study, FIST is a feasible option in participating institutions. All FIST residents, regardless of track, met requirements for ABS Board eligibility, despite modifications to rotations and case experience. Future studies will assess the impact of FIST on ABS exam results and fellowship success.

A novel, simplified, externally validated staging system for truncal/extremity soft tissue sarcomas: An analysis of the US Sarcoma Collaborative database: JOHNSON et al.

The 8th edition AJCC staging system for truncal/extremity soft tissue sarcoma (STS) offers significant changes from the 7th. However the complexity of both limits their clinical utility.

Abstract 4150: Blockade of CXCR2 mediated granulocytic MDSC recruitment synergizes with CCR2 inhibition of inflammatory monocytes and restores anti-tumor immunity in pancreatic adenocarcinoma

Introduction: Pancreatic cancer (PC) is characterized by a dense tumor stroma with a heavy leukocytic infiltrate, comprised predominately of immunosuppressive bone marrow (BM) derived cells. We have previously demonstrated in a phase Ib clinical trial that CCR2 inhibition (CCR2i) prevents inflammatory monocyte (IM) recruitment from the BM and results in a significant reduction of tumor associated macrophages (TAM) and an increase in treatment efficacy. However, granulocytic myeloid derived suppressor cells (G-MDSC) remain in the tumor microenvironment (TME) following CCR2i. Herein, we explored the impact of targeting G-MDSC recruitment to PC tumors both alone and in combination with CCR2i. Methods: Human BM, blood, and tumor was collected under an IRB approved protocol. A tissue microarray (TMA) from resected PC patients was analyzed for immune infiltrate. Mice were injected orthotopically with 2.5×106 syngeneic PC cells. CXCR2 and CCR2 inhibitors (Tocris) were given twice daily. Tumor growth was assessed and specimens obtained for analysis by flow cytometry, RNAseq, and IHC. Results: Human PC overexpresses CXCL5 and CXCL8, corresponding with an abundance of tumor infiltrating CXCR2+ G-MDSC. Furthermore, the ratio of CD8 to G-MDSC correlates with survival in human PC patients. In an orthotopic murine model that recapitulates human disease, ΣCXCL ligands were also increased. Either Ly6G depletion or targeted blockade with a CXCR2 inhibitor decreased G-MDSC and reduced tumor burden. Intriguingly, blockade of IM from the BM did not reduce G-MDSC and paradoxically resulted in a modest increase in this population within the tumors from human patients following CCR2i. Thus, we explored the combination of CCR2/CXCR2 blockade both with and without FOLFIRINOX chemotherapy. This resulted in a synergistic impact when both BM derived populations were targeted and dual therapy was further enhanced by FOLFIRINOX. RNAseq analysis of tumors following monotherapy or dual inhibition revealed alterations in the TME favoring an anti-tumor immune response. To test the hypothesis that this effect was mediated by restoration of anti-tumor immunity we analyzed the tumor infiltrating lymphocyte (TIL) populations and found a significant increase in the relative and absolute numbers of CD8+ and C4+ TIL. Analysis of the activation status of these cells demonstrated an increase in effector CD8+ T-cell phenotype (IFNγ+, CD69+, CD44+). Using Nur77GFP T-cell receptor reporter mice, we showed an increase in GFP expressing CD8+ TIL following dual blockade. CD8 depletion resulted in a loss of therapeutic efficacy of myeloid blockade, further confirming our hypothesis. Conclusion: These findings suggest that combinatorial blockade strategies preventing tumor infiltration by myeloid cells may restore anti-tumor immunity in PC. Citation Format: Timothy M. Nywening, Brian A. Belt, Roheena Z. Panni, Darren Cullinan, Dominic E. Sanford, Ryan C. Fields, William G. Hawkins, David G. DeNardo, William E. Gillanders, Peter Goedegebuure, David C. Linehan. Blockade of CXCR2 mediated granulocytic MDSC recruitment synergizes with CCR2 inhibition of inflammatory monocytes and restores anti-tumor immunity in pancreatic adenocarcinoma. [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 4150.

Abstract 2350: Vaccination enhances anti-tumor immunity in ovarian cancer following repolarization of the tumor microenvironment with CCR2 blockade

Introduction: Ovarian cancer (OC) expresses the tumor associated antigen mesothelin and contains a relative abundance of T-cells. However, ovarian cancer is also infiltrated by immunosuppressive tumor associated macrophages (TAM) that dominate the tumor microenvironment (TME). The CCL2/CCR2 chemokine axis is co-opted by various human malignancies to facilitate the recruitment of bone marrow (BM) derived inflammatory monocytes (IM) to the TME where they become immunosuppressive TAMs. Herein, we explore the rationale for combination of a CCR2 inhibitor (CCR2i) with a mesothelin peptide vaccine. Methods: Monocyte counts were obtained from preoperative CBCs under IRB approval. Mice were vaccinated with a dual eight-mer peptide (50 nM/vaccination) on days 0 and 7 with an irradiated peptide pulsed dendritic cell boost on day 14. Mice were challenged with 4 million syngeneic OC cells (ID8) on day 15. CCR2 inhibitor (Tocris) and CCR2 KO mice were used. Results: Preoperative monocyte counts of human ovarian cancer patients were stratified into low (>1 SD below mean), mid (within 1 SD of mean), and high (>1 SD above the mean) groups. Patients with a high monocyte count (n = 15) had a significantly decreased median survival of 1.2 years compared to 4.8 years in the low monocyte group (n = 15). The mid group (n = 69) had a median survival of 3.5 years (p 0.001). The hazard ratio between the low and high groups was 0.24 (0.05-0.39). Flow cytometry of peripheral blood from these patients demonstrated that the majority of these monocytes were CCR2+ inflammatory monocytes. Human OC overexpresses CCL2 compared to normal ovarian tissue and analysis of the TME from resected human OC patients revealed an abundance of CCR2+ TAM, which greatly outnumbered tumor infiltrating lymphocytes (TIL). In a murine ID8 tumor model, which recapitulates features of human OC, CCR2i prevented IM egress from the bone marrow with a resultant decrease in TAM at the primary tumor site. Furthermore, there was an increase in TIL infiltrate following CCR2 blockade. Addition of vaccine to CCR2i caused an improvement of effector to suppressor ratio and prolonged survival compared to vaccine (p = 0.02) or CCR2i alone (p = 0.02) and control (p Conclusion: Thus far vaccination has not provided durable patient responses in OC. Therapies targeting the immunosuppressive TME are an attractive treatment modality to enhance vaccination and facilitate anti-tumor immunity in OC. Citation Format: Darren Cullinan, Pratibha Binder, Timothy Nywening, Ivy Wilkinson-Ryan, Brian Belt, Peter Goedegebuure, David Linehan, Matthew Powell, William Hawkins. Vaccination enhances anti-tumor immunity in ovarian cancer following repolarization of the tumor microenvironment with CCR2 blockade. [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 2350.