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Prehabilitation is well established for improving outcomes in cancer surgery. Combining prehabilitation with neoadjuvant treatments may provide an opportunity to rapidly initiate cancer-directed therapy while improving functional status in preparation for local consolidation. In this proof-of-concept study, we analyzed non-small-cell lung cancer patients who underwent simultaneous prehabilitation and neoadjuvant therapy.We retrospectively analyzed all patients who underwent neoadjuvant treatment for non-small-cell lung cancer followed by curative intent surgery between 2015 and 2021. Patients who were screened for the prehabilitation program were identified. The screening included assessment of physical performance, nutritional status, and signs of anxiety and depression.We identified a total of 141 patients who underwent neoadjuvant therapy. Twenty patients were screened to undergo a prehabilitation program. Four patients did not complete the exercise program (1 surgical intervention too soon, 1 drop-out after the first session, and 2 patients were deemed fit without intervention). The postoperative median length of stay was 2 days (range 1-18). Patients improved their 6-minute-walk test despite undergoing neoadjuvant treatment by a mean of 33 meters (± 50, P = .1). Self-reported functional status (DASI) showed significant improvement by a mean of 10 points (± 11, P = .03), and HADS-anxiety-score was significantly reduced after the prehabilitation program by a mean of 1.5 points (± 1, P = .005).Neoadjuvant prehabilitation therapy is feasible and associated with encouraging results. The performance of all measures remains a logistic challenge. With multimodal strategies for lung cancer treatment becoming key to optimal outcomes, neoadjuvant prehabilitation therapy is a concept worthy of prospective multi-center evaluation.

Adenocarcinoma of the proximal stomach is the fastest rising malignancy in North America. It is commonly associated with peritoneal accumulation of malignant ascites (MA), a fluid containing cancer and inflammatory cells and soluble proteins. Peritoneal metastasis (PM) is the most common site of gastric cancer (GC) progression after curative-intent surgery and is the leading cause of death among GC patients.Using a panel of gastric adenocarcinoma cell lines (human: MKN 45, SNU-5; murine: NCC-S1M), we demonstrate that prior incubation of GC cells with MA results in a significant ( 1.7-fold) increase in the number of cells capable of adhering to human peritoneal mesothelial cells (HPMC) (p 0.05). We then corroborate these findings using an ex vivo PM model and show that MA also significantly enhances the ability of GC cells to adhere to strips of human peritoneum (p 0.05). Using a multiplex ELISA, we identify MIF and VEGF as consistently elevated across MA samples from GC patients (p 0.05). We demonstrate that agents that block the effects of MIF or VEGF abrogate the ability of MA to stimulate the adhesion of GC cells to adhere to human peritoneum and promote both ex vivo and in vivo metastases.Agents targeting MIF or VEGF may be relevant to the treatment or prevention of PM in GC patients.

332 Background: Docetaxel-based therapy is currently the most effective neoadjuvant regimen for locally advanced gastroesophageal adenocarcinoma. However, prior trials were primarily designed to target gastric cancer. As survival associated with taxane-based chemotherapy in esophageal adenocarcinoma (EAC) is less well described, we sought to review our experience with docetaxel based therapy as a neoadjuvant approach for EAC. Methods: A single centre retrospective review of a prospectively maintained upper GI cancer surgical database was performed (2008-2021). Patients with EAC undergoing curative intent neoadjuvant therapy with two similar docetaxel-based regimens (DCF - Docetaxel/Cisplatin/5FU or FLOT - 5FU, Leucovorin, Oxaliplatin, and Docetaxel) followed by en-bloc resection were included. Gastric/EGJ type III tumors were excluded. Clinicopathological data and overall survival (OS) were recorded and stratified by chemotherapy type (DCF vs FLOT). Data are presented as median (+/-SD). Mann-Whitney U tests for continuous or Fischer exact test for categorical variables determined significance. Kaplan-Meier curves compared OS. Results: 225 EAC patients were identified: Age 64.2 (±10.3) and tumor location was Esophageal/EGJ I/EGJ II (106/33/86). Clinical stage was cT3-4 in 201/225 (89.3%) and most were cN+ (200/225: 88.9%). There were 121 (53.7%) treated with neoadjuvant DCF whilst 104 (46.3%) received FLOT. All pre-op cycles were completed in 181 (80.4%) and did not differ between DCF and FLOT (81.8% vs 78.8% - NS). Operative procedure included Ivor Lewis in 188 (83.5%), left thoraco-abdominal esophagectomy in 20 (8.8%), and McKeown in 17 (7.5%) and also did not differ between DCF and FLOT. In terms of pathologic outcomes, the median lymph node yield was 35 (±16) of which 3.6±5.8 were positive). There was no difference when comparing between regimens. There were 134 (59.6%) patients ypN1-3 (DCF=76(62.8%) vs FLOT=58(55.8%)- NS). An R0 resection was achieved in 210 (93%) patients (DCF=92.6% vs FLOT=94.2%- NS). Complete pathological response (no residual cancer) was found in 21 patients (9.3%) (DCF=14(11.6%) vs FLOT=7(6.7%) – NS). At follow up of 41(±36) months (DCF= 52±41 vs FLOT=27±23 - p

333 Background: Neoadjuvant therapy followed by en bloc surgical resection affords the highest rates of survival from locally advanced esophageal cancer (LAEC) and represents the standard of care. However, patients of advanced age may not be offered this approach due to concerns over toxicity/tolerability. The outcomes of different treatment modalities for patients aged 80 and above with LAEC are not well described. Methods: A retrospective, single center, cohort analysis was performed on a prospectively maintained comprehensive esophageal cancer database. Between 2010-20, all patients ≥80yrs with locally advanced esophageal/GEJ cancer (cT2-4a, Nany, M0) were identified and outcomes stratified according to the following treatment categories: Neoadjuvant chemotherapy (nCT) or chemoradiotherapy (nCRT) followed by surgery; definitive CRT (dCRT); upfront surgery; palliative CT or RT; or best supportive care (BSC). Data presented as median(range). Univariate analysis used for clinicopathological data (*p

Despite advances in cancer treatment, metastasis remains today the main cause of cancer death. Local control through complete surgical resection of the primary tumor continues to be a key principle in cancer treatment. However, surgical interventions themselves lead to adverse oncologic outcomes and are associated with significantly increased rates of metastasis. Neutrophils through release of neutrophil extracellular traps (NETs) in response to infections were shown to be able to capture circulating cancer cells, and in doing so, support the development of metastatic disease. To be able to intervene on this process, understanding the exact molecular nature of these mechanisms is crucial. We therefore hypothesize and demonstrate that β1-integrin is an important factor mediating the interactions between circulating tumor cells and NETs. We show that β1-integrin expression on both cancer cells and NETs is important for the adhesion of circulating tumor cells to NETs both in vitro and in vivo. Using a murine model of intra-abdominal sepsis to mimic the postoperative inflammatory environment, we show that β1-integrin expression is upregulated in the context of inflammation in vivo. Ultimately, we show that this increased early cancer cell adhesion to NETs in vivo and this effect is abrogated when mice are administered DNAse 1. Our data therefore sheds light on the first molecular mechanism by which NETs can trap circulating tumor cells (CTCs), broadening our understanding of this process.

e20545 Background: Prehabilitation is well established for improving outcomes in cancer surgery. Combining prehabilitation with neoadjuvant treatments may provide an opportunity to rapidly initiate cancer-directed therapy while improving functional status in preparation for local consolidation. In this proof-of-concept study, we analyzed non-small-cell lung cancer patients who underwent simultaneous prehabilitation and neoadjuvant therapy. Methods: We retrospectively analyzed all patients who underwent neoadjuvant treatment for non-small-cell lung cancer followed by curative intent surgery at the McGill University Health Center between 2015-2020. Patients who were screened for the prehabilitation program were identified. Screening included assessment of physical performance, nutritional status and signs for anxiety and depression. 6-minute-walk test was used as a functional outcome parameter of prehabilitation. Results: We identified a total of 93 patients who underwent neoadjuvant therapy. Of these, 12 patients were screened to undergo a prehabilitation program. For 1 patient surgical intervention was too soon to complete the program, 1 patient dropped out after the first and another patient was deemed fit to undergo surgery without intervention. Thus, 9 patients completed full neoadjuvant prehabilitation therapy. Postoperative median length of stay was 2 days (IQR 1-5) and there were no mortalities. We found major complications in 1 patient and minor complications (prolonged air leak) in 2 cases. Patients improved their 6-minute-walk test despite undergoing neoadjuvant treatment by a mean of 35 meters (SD 39). Conclusions: Neoadjuvant prehabilitation therapy is feasible and associated with encouraging results. The performance of all measures remains a logistic challenge. With multimodal strategies for lung cancer treatment becoming key to optimal outcomes, neoadjuvant prehabilitation therapy is a concept worthy of prospective multi-center evaluation.

Introduction Surgery is essential for cure of early-stage non-small cell lung cancer (NSCLC). Rates of postoperative bacterial pneumonias, however, remain high, and clinical data suggests that post-operative infectious complications confer an increased risk for metastasis. Toll-like receptors (TLRs) mediate the inflammatory response to infection by recognizing evolutionarily conserved bacterial structures at the surface of numerous pulmonary cell types; yet, little is known about how host TLR activation influences NSCLC metastasis. TLR4 recognizes gram-negative bacterium lipopolysaccharide activating the innate immune system. Methods C57BL/6 and TLR4 knockout murine airways were inoculated with Escherichia coli or lipopolysaccharide. Hepatic metastasis assays and intravital microscopy were performed. Bronchoepithelial conditioned media was generated through coincubation of bronchoepithelial cells with TLR4 activating Escherichia coli or lipopolysaccharide. Subsequently, H59 NSCLC were stimulated with conditioned media and subject to various adhesion assays. Results We demonstrate that gram-negative Escherichia coli pneumonia augments the formation of murine H59 NSCLC liver metastases in C57BL/6 mice through TLR4 activation. Additionally, infected C57BL/6 mice demonstrate increased H59 NSCLC in vivo hepatic sinusoidal adhesion compared with negative controls, a response that is significantly diminished in TLR4 knockout mice. Similarly, intratracheal injection of purified TLR4 activating lipopolysaccharide increases in vivo adhesion of H59 cells to murine hepatic sinusoids. Furthermore, H59 cells incubated with bronchoepithelial conditioned medium show increased cell adhesion to in vitro extracellular matrix proteins and in vivo hepatic sinusoids through a mechanism dependent on bronchoepithelial TLR4 activation and interleukin-6 secretion. Conclusion TLR4 is a viable therapeutic target for NSCLC metastasis augmented by gram-negative pneumonia.

The best surgical approach for tumors of the proximal stomach remains controversial. For proximal gastrectomy (PG), the evidence regarding quality of life (QoL) and functional outcomes is controversial. Moreover, there are limited data from non-Asian settings. All patients who underwent PG from September 2005 to July 2013 were identified from an institutional database. Demographic, perioperative and pathologic characteristics were retrieved. Symptom scores (0 = best/4 = worst) for reflux symptoms, dysphagia and validated QoL metrics (FACT scale, where a higher score is better) were assessed during early and late follow-up. Eligible patients for analysis were those with no evidence of recurrence. Of 465 upper gastrointestinal cancer resections, 50 were PG for adenocarcinoma (42; 84 %), neuroendocrine carcinoma (5; 10 %) or other pathologies (3; 6 %). R0 resection was achieved in 44 (89.8 %) of 49 patients with malignant tumors. Median lymph node collection was 32 (range 7–57). QoL scores did not differ from preoperative to early follow-up but increased compared to both at late follow-up [preoperative, 125 (interquartile range 105–140); early follow-up, 122.5 (97–142); late follow-up, 147 (132–159); p

We have previously identified that Neutrophil Extracellular Traps (NETs) are an important contributing factor to the metastatic process. However, the underlying molecular mechanisms by which NETs facilitate metastasis remain unclear. Using mass spectrometry, amongst 583 distinct proteins, we identified CEACAM1 (CC1) in isolated human NETs and we sought to determine whether CC1 has a role in NET-mediated cancer metastasis. The presence of CC1 on NETs was confirmed by immunofluorescence. In vitro static adhesion of human colon cancer HT29 cells to isolated purified human NETs was reduced by 50% using a function blocking antibody against human CC1, but not isotype control, an effect equal to NET degradation with DNAse. Adhesion of murine colon cancer MC38-CC1L cells to C57BL/6 mouse neutrophils stimulated to produce NETs with phorbol myristate acetate (PMA) was increased 3-fold compared to untreated neutrophils, an effect that was completely attenuated with DNAse. PMA stimulation of neutrophils isolated from Ceacam1−/− knockout (KO) mice did not increase adhesion to MC38-CC1L cells. Using a parallel plate flow chamber, a physiologically relevant adhesion model under the shear conditions encountered in liver sinusoids, we flowed Lewis lung carcinoma (C10) cancer cells over neutrophils isolated from C57BL/6 or Ceacam1−/− KO mice. PMA-induced NET formation in C57BL/6 mouse neutrophils increased cancer cell adhesion 5-fold, an effect again completely attenuated by DNAse or use of neutrophils from Ceacam1−/− KO mice. Using a transwell chamber, MC38-CC1L cancer cells exposed to NET-stimulated Ceacam1−/− KO neutrophils had a 50% decrease in migration, compared to those exposed to NETs from C57BL/6 neutrophils. In order to delineate the role of CC1 in NET-related in vivo adhesion of cancer cells to liver sinusoids, we performed a series of neutrophil depletion and re-infusion experiments. C57BL/6 mice, depleted of neutrophils with anti-GR1 24 hrs prior, were re-infused with neutrophils isolated from C57BL/6 or Ceacam1−/− KO mice. This was followed by injection of MC38-CFSE labelled cells and hepatic intravital microscopy was used to quantify in vivo cancer cell adhesion and migration. PMA-stimulated C57BL/6 neutrophils prior to re-infusion was associated with a two-fold increase in adhesion compared to PMA-stimulated Ceacam1−/− KO neutrophils, an effect that was completely attenuated using DNAse. Our data support the notion that CEACAM1 is, at least in part, responsible for the increased cancer cell migration mediated by Neutrophil Extracellular Traps. We have thereby identified NET-associated CEACAM1 as a putative therapeutic target to prevent the metastatic progression of cancer cells. Citation Format: Phil Vourtzoumis, Rashmi Seth, Roni Rayes, Sara Najmeh, Jonathan Cools-Lartigue, Betty Giannias, France Bourdeau, Nicole Beauchemin, Simon Rousseau, Richard Blumberg, Jonathan D. Spicer, Lorrenzo Edwin Ferri. The role of CEACAM1 in neutrophil extracellular Trap mediated cancer metastasis. [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 LB-078.

INTRODUCTION: Strong clinical evidence demonstrates a link between acute bacterial infection and metastasis. We and others have shown that activation of certain membrane pattern recognition receptors (PRRs), such as Toll-like receptors, on cancer cells can be implicated in this process. There is emerging data that a cytoplasmic PRR, the nucleotide oligomerization domain receptor 1 (NOD1), may also play an important and complementary role in the immune response to bacterial infection, however its role in cancer progression is entirely unknown. We sought to determine the influence of NOD1 activation on metastasis. METHODS: NOD1 expression in human and murine colon (HT29, MC38) and lung (A549, H59) cancer cells were confirmed using flow cytometry (FC) and immunoblotting (WB). A series of in vitro and in vivo functional assays, including adhesion, migration and hepatic intravital microscopy (IVM), was conducted to assess the effect of NOD1 activation and inhibition. C12-iE-DAP, a highly selective NOD1 ligand derived from gram-negative bacterial wall, was used to simulate NOD1-activation under infectious condition. ML130, a specific NOD1 inhibitor, was used to block C12-iE-DAP activation. Stable knockdown (KD) of NOD1 in HT29 and A549 cells were constructed with shRNA lentiviral transduction and the functional assays were thus repeated. The predominant signaling pathway, downstream cytokines and cell adhesion molecules of NOD1-activation were identified using WB in the presence of kinase inhibitors. RESULTS: WB and FC showed abundant NOD1 expression in all tested cell lines. Cancer cells stimulated with C12-iE-DAP doubled the in vitro adhesion to collagen I, IV and fibronectin, as well as in vitro migration, an effect completely attenuated with ML130 inhibition and NOD1 knockdown. Using a murine hepatic adhesion assay under IVM, we observed a doubling in the amount of in vivo capture of cancer cells within hepatic sinusoids of C57BL6 mice in the C12-iE-DAP group compared to control, an effect again abrogated by ML130 inhibition and NOD1 knockdown. Immunoblotting using HT29 cells under a panel of kinase inhibitors revealed that NOD1 activation is p38 dependent. Subsequently, the use of p38 inhibitor, BIRB0796, abolished C12-iE-DAP mediated adhesion to collagen I and fibronectin. Using human cytokine quantification array, we determined that NOD1 activation led to increase in IL-8, 15, MCP1 and RANTES, all of which have been shown to link to cancer progression or to chemoattract neutrophils, which we have recently shown to mediate metastasis. CONCLUSION: Our data demonstrate that NOD1 activation by the gram-negative bacterial wall component, C12-iE-DAP is important in enhancing the metastatic potential of cancer cells, and its mechanism is dependent on p38 activation and the upregulation of downstream cytokines. This is the first study to implicate NOD1 in metastasis, and thus identify this receptor as a putative therapeutic target. Citation Format: Henry Jiang, Sara Najmeh, Julie Berube, Arielle Leone, Paul Savage, Betty Giannias, France Bourdeau, Simon Rousseau, Morag Park, Lorenzo Edwin Ferri. NOD1 augments cancer cell metastatic potential through p38 MAP kinase activation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3162. doi:10.1158/1538-7445.AM2015-3162

Introduction: Post-operative infectious complications in cancer patients confer an increased risk of death from distant metastasis, and activated neutrophils appear to be central cellular players in this phenomenon. We have previously demonstrated that Neutrophil extracellular traps (NETs), composed of extracellular neutrophil-derived DNA webs, are intricately implicated in this process by directly binding and capturing circulating tumor cells. However, the mechanisms mediating this interaction remain completely unexplored. We hypothesized that tumor cells adhere to NETs specifically through interactions between neoplastic and neutrophil-derived β 1 integrins. Methods: In vitro, NETs isolated from phorbol myristate acetate (PMA) activated neutrophils from healthy volunteers were plated on cover slips and human lung or colorectal cancer (A549 or HT29) adhesion assessed. In some experiments, cancer cell adhesion was assessed in the presence of DNAse, RGD peptide, or anti- β 1 integrin antibody (4B4). In vivo, cecal ligation and puncture (CLP) was used to induce NET formation and A549 cells (siRNA anti- β 1 integrin transfected or control) were injected via the intrasplenic route to assess real time single cell migration by hepatic intravital microscopy. Results: Adhesion of cancer cells to NET isolates was decreased 2-3 fold in the presence of DNAse and proteinase K, indicating the possibility of protein-protein interactions. Pretreatment of malignant cells with RGD peptide, known to inhibit integrin mediated adhesion, similarly resulted in a 2-3 fold decrease in tumor cell adhesion compared to isolated NETs alone. Adhesion of A549 and HT29 cells to NET isolates after pretreatment with function blocking anti- β 1 integrin antibody was 1/3 that of untreated cells. Following PMA stimulation, β 1 integrin staining co-localized within extracellular DNA but was absent in unstimulated neutrophils. Adhesion in vivo was reduced 2 fold after siRNA knockdown of β 1 integrin in A549 cells 24 hours following CLP compared to untreated cells. Hepatic intravital microscopy after CLP demonstrates widespread NET deposition demonstrated by DNA and histone H2A.X deposition. NETs were found to stain positive for β 1 integrin. Beta 1 integrin staining was increased after CLP compared to sham, and was abrogated by neutrophil depletion. Conclusions: This study is the first to implicate β 1 integrin mediated interactions as a mechanism for tumor-NET adhesion. Cell surface expression of β 1 integrins on tumor cells was necessary for tumor cell adhesion to NETs both in vitro and in vivo. Analysis of NETs both in vitro and in vivo demonstrated β 1 integrin expression, implicating integrin hetero-dimerization as a possible adhesive mechanism. Citation Format: Sara Najmeh, Jonathan Cools-Lartigue, Stephen Gowing, Jonahan Spicer, Braedon McDonald, Betty Giannias, France Bourdeau, Paul Kubes, Lorenzo Edwin Ferri. Neutrophil extracellular traps sequester circulating tumor cells via beta 1 integrin mediated interactions. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-258. doi:10.1158/1538-7445.AM2014-LB-258