Spatiotemporal immune dynamics in lung cancer progression and treatment.

Background: Lung cancer is a multifactorial disease with a heterogeneous tumor group that hampers diagnostic and therapeutic approaches, as well as understanding of the processes that underlie its pathogenesis. Current research efforts are focused on examining alterations in the tumor microenvironment, which may affect the pathogenesis and further malignant progression in lung cancer. The aim of this study was to investigate changes in the levels of biomarkers involved in the lung tumor microenvironment and their diagnostic utility in differentiating lung cancer subtypes and stages. Methods: This study comprised 112 lung cancer patients, 50 with adenocarcinoma, 35 with squamous cell carcinoma, 13 with other non-small cell lung carcinoma subtypes, and 14 with other lung neoplasms than non-small cell lung carcinoma. Tumor markers (CEA, CYFRA 21-1, and NSE) were measured in the patients’ sera and plasmas, along with IL-6, TNF-α, SAA1, CRP, MMP-2, MMP-9, glucose, lactate, and LDH, utilizing enzyme-linked immunosorbent assays, enzyme immunoassays, and automated clinical chemistry and turbidimetry systems. The results were statistically analyzed across patient groups based on the subtype and stage of lung cancer. Results: Glucose concentrations showed statistically significant (p < 0.05) differences both between lung cancer subtypes and stages, with the highest levels in patients with other lung neoplasms (me = 130.5 mg/dL) and in patients with stage IIB lung cancer (me = 132.0 mg/dL). In patients with advanced lung cancer, IL-6 and LDH had considerably higher concentration and activity. There was also a significant positive correlation between IL-6 and MMP-9 in adenocarcinoma and SqCC, with correlation coefficients of 0.53 and 0.49, respectively. The ROC analyses showed that the best single biomarkers for distinguishing adenocarcinoma from squamous cell carcinoma are glucose, CRP, and CYFRA 21-1; however, their combination did not significantly improve sensitivity, specificity, and the AUC value. The combinations of IL-6, glucose, LDH and CEA, IL-6, SAA1, MMP-9, and lactate can distinguish patients with stage IIB lung cancer from those with stage IIA with 100% sensitivity, 100% specificity, and with an AUC value of 0.8333 and 1.0000, respectively, whereas the combination of CEA, IL-6, and LDH can identify patients with stage IIIA lung cancer from those with stage IIB with 72.73% sensitivity, 94.44% specificity, and an AUC value of 0.8686. Conclusion: There is a link between biomarkers of tumor microenvironment changes and tumor markers, and combinations of these markers may be clinically useful in the differential diagnosis of adenocarcinoma and squamous cell carcinoma, as well as lung cancer stages IIB and IIA, and IIIA and IIB.

EUS staging of primary lung carcinoma: are we ready for it?

Background: The use of immunotherapy to treat lung cancer is becoming increasingly common, highlighting the importance of the immune system in the lung tumor microenvironment. The lungs are host to a variety of immune cell subsets, including eosinophils (Eo), which are a population of innate immune cells that exert cytotoxic effector functions through the release of secretory granules and participate in tissue homeostasis and immunity. Despite the presence of Eo in solid tumors and their prevalence in the lung, the role of Eo in lung cancer is both controversial and largely unexplored. The Bennewith lab has previously found that mice with elevated lung Eo have decreased tumor growth in a model of breast cancer lung metastasis. We hypothesize that Eo play a protective role in lung cancer progression. Methods: In collaboration with Dr. Kelly McNagny (UBC), we used IL-5Tg transgenic mice that overexpress IL-5 and have a systemic expansion of Eo, ddGATA transgenic mice which are Eo-deficient, and ddGATA/IL-5Tg double-transgenic mice (excess IL-5 but no Eo) to study lung cancer. Lewis Lung carcinoma (LLC) cells were injected intravenously (IV) to seed the lungs. After three weeks, we harvested lungs and used flow cytometry to quantify immune cell subsets in the lungs. Additionally, we used clonogenic assays and histology to quantify lung tumor growth. Results: We confirmed that naive ddGATA and ddGATA/IL-5Tg mice have no lung Eo. In contrast, IL-5Tg mice have a 100-fold expansion of Eo in the lungs, and these Eo express higher levels of the Eo activation marker CD11b compared to wild-type (WT) mice. Naive IL-5Tg and ddGATA/IL-5Tg mice had an increased proportion of lung B-1 B cells, as well as an increase in the expression of the apoptosis-inducing cell surface molecule FasL. The absence of Eo in ddGATA mice did not impact lung colonization of LLC cells. Though there was a substantial expansion of Eo in the lungs of IL-5Tg mice compared to WT mice, there was no change in the number of lung-infiltrating Eo three weeks after IL-5Tg and WT mice were injected IV with LLC cells. IL-5Tg mice injected IV with LLC cells had an increase in the total number of lung-infiltrating Bconv and B-1 B cells compared to naive mice, whereas there was no change in B cell subsets between naive and LLC IV injected WT mice. Conclusions: Though Eo may play an antitumorigenic role in the presence of excess IL-5, the absence of Eo in ddGATA mice did not result in an increase in lung tumor burden. This suggests that Eo need to be activated and expanded to exert an antitumorigenic effect, or that the expansion of B cells in IL-5Tg mice is responsible for the decrease in lung tumor growth in IL-5Tg mice relative to WT mice. Illuminating the specific roles Eo and B cells play in lung cancer progression will allow us to better understand the interplay between host immune cells and malignant cells and could reveal new avenues of cancer immunotherapy development. Citation Format: Rachel A. Cederberg, Alvina So, Elizabeth Franks, Jenna Collier, Brennan J. Wadsworth, Michael R. Hughes, Kelly M. McNagny, Kevin L. Bennewith. The role of eosinophils in the lung tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A75.

With over a million deaths every year around the world, lung cancer is found to be the most recurrent cancer among all types. Nonsmall cell lung carcinoma (NSCLC) amounts to about 85% of the entire cases. The other 15% owes it to small cell lung carcinoma (SCLC). Despite decades of research, the prognosis for NSCLC patients is poorly understood with treatment options limited. First, this article emphasises on the part that tumour microenvironment (TME) and its constituents play in lung cancer progression. This review also highlights the inflammatory (pro- or anti-) roles of different cytokines (ILs, TGF-β, and TNF-α) and chemokine (CC, CXC, C, and CX3C) families in the lung TME, provoking tumour growth and subsequent metastasis. The write-up also pinpoints recent developments in the field of chemokine biology. Additionally, it covers the role of extracellular vesicles (EVs), as alternate carriers of cytokines and chemokines. This allows the cytokines/chemokines to modulate the EVs for their secretion, trafficking, and aid in cancer proliferation. In the end, this review also stresses on the role of these factors as prognostic biomarkers for lung immunotherapy, apart from focusing on inflammatory actions of these chemoattractants.

Despite decades of research, non-small cell lung carcinoma (NSCLC) remains one of the world's deadliest cancers. Approximately half of NSCLC cases are classified as lung adenocarcinomas (ADCA), in which KRAS, EGFR, and TP53 mutations are the predominant genetic drivers. Although patients with EGFR mutations are initially responsive to targeted therapies, drug resistance uniformly occurs within one year. KRAS mutant ADCA have proven even more intractable, as scant progress has been made in the development of therapeutics for this patient population. Recent breakthroughs in the field of immunotherapy have offered new hope for the management of NSCLC, but the clinical success of immunomodulatory agents will depend on a strong foundational knowledge of the immune cells that comprise the ADCA microenvironment. Activating mutations in KRAS and EGFR induce the aberrant function of numerous signaling pathways that may influence the host immune response in different ways. However, the discrete impact of KRAS and EGFR mutations on the immune composition of ADCA remains largely undefined. To address this question, we profiled the tumor microenvironments (TME) of four mouse models of ADCA: adenoviral Cre-infected KrasLSL-G12D and KrasLSL-G12D;Trp53Fl/Fl mice, as well as TetO-EgfrL858R;Ccsp-rtTA and TetO-EgfrL858R;T790M;Ccsp-rtTA mice. Lung specimens from tumor-bearing and control animals (n ≥ 5 per cohort) were harvested at multiple time points and subjected to histological assessment, gene expression analysis by qRT-PCR, and flow cytometric immunophenotyping, in which 23 markers were used to identify 12 unique cell types. The Kras subtype exhibited robust inflammation and expansion of both the lymphoid and myeloid cell compartments. In particular, significant increases were observed in the number of CD8+ T cells (3-fold), B cells (2.5-fold), and macrophages (16-fold). Although loss of Trp53 promoted tumor growth and malignancy, it had little effect on cytokine expression or immune cell recruitment into the Kras TME. In contrast, while EgfrL858R mutations produced similar total levels of inflammation, leukocyte recruitment into the Egfr TME was largely restricted to macrophage expansion (increased 8-fold). Introduction of the T790M resistance mutation to Egfr-mutant animals further amplified the macrophage dominant signal (14-fold). Expression of the cytokine IL-6 was correspondingly elevated in the Egfr but not Kras subtypes, a finding replicated in EGFR-transfected primary human endobronchial cells. Most notably, CD8+ cytotoxic T cell numbers were unaltered at all time points examined in the Egfr animals, a finding which raises concerns about the efficacy of most currently developed immune checkpoint blockers in EGFR patients. In summary, KRAS and EGFR mutations give rise to distinct immune responses that may require oncogene-specific immunotherapy regimens. Citation Format: Stephanie E. Busch, Mark L. Hanke, Kyoung Hee Kim, Julia Kargl, A. McGarry Houghton. EGFR and KRAS activation generate discrete inflammatory responses within the lung tumor microenvironment. [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 3181. doi:10.1158/1538-7445.AM2015-3181

Background: Immune cells within the tumor microenvironment (TME) play a vital role in regulating tumor progression. Therefore, immunotherapies that elicit anti-tumor responses are of great interest for the treatment of various cancers. Macrophages are a current immune cell type of interest due to the ability to polarize into anti-tumor (M1) and pro-tumor (M2) phenotypes. The density and phenotype of macrophages within the tumor and TME have been linked to prognosis in multiple types of solid tumors. Our hypothesis is that non-small cell lung carcinoma (NSCLC) patients with high immune infiltration and greater amounts of anti-tumor immune cells within the tumor compartment will have an increased time of survival compared to cancers with immune excluded or immune desert environments. Methods: One NSCLC tumor microarray (TMA) containing primary tumors, metastases, and normal tissue were stained via multiplex immunofluorescence (mIF) for 6 different immune markers: CD3, CD8, CD56, CD68, CD163, and PD-L1. The stained TMAs were analyzed utilizing Flagship Biosciences’ proprietary image analysis platform. Machine learning algorithms used cellular features to stratify cells as belonging to either the tumoral or stromal compartment. Core level expression data was pulled and represented on a whole-cohort basis. All staining and image analysis outputs were reviewed by a board-certified, MD pathologist. Kaplan-Meier curves were generated based on survival data in relation to the low, medium, and high expression of CD68 and/or CD163 in the whole tissue, as well as tumor and stromal compartments. Patients were also stratified into low (1 and 2) and high (3 and 4) cancer stages. Results: There is a correlation between patient survival and the presence or absence of macrophage markers CD68 and CD163. Specifically, lower levels of CD68+CD163- cells within the tumor and stroma compartments correlate with an increase in patient survival. There is also a correlation of a high level of expression of CD163+, CD68+CD163-, or CD68+CD163+ cells with increased patient survival in high stage patients compared to low stage patients. Specifically in high stage NSCLC patients, a higher level of CD163+ expression correlates with a reduced lifespan compared to patients with medium and low expression. Conclusion: Data generated through Flagship Biosciences’ image analysis platform showed a strong relationship between immune cell presence and localization with NSCLC patient survival. Altering the immune cells within the tumor to an anti-tumor immune environment could increase patient survival times. Further, utilizing Flagship Biosciences’ image analysis software to understand cancer immune microenvironments should be further utilized to aid in diagnosis and treatment decisions. Citation Format: Dannah Miller, Kate Hieber, Will Paces, Huong Nguyen, Adam Beharry, Roberto Gianani. Macrophage biomarkers CD68 and CD163 correlate with NSCLC patient survival [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 1720.

<h3>Background</h3> Cancer-associated fibroblasts (CAFs) are a major component of the non-small cell lung cancer (NSCLC) tumor microenvironment (TME).^1–4 Recent studies indicate that CAFs play a role in generating a CD8+T...

BACKGROUND:Abdominal aortic aneurysm (AAA) is characterized by sterile inflammation, immune cell infiltration, and stromal remodeling that progressively weaken the aortic wall, leading to life-threatening aortic rupture. The molecular mechanisms and spatial organization of immune–stromal interactions in human tissue are poorly understood, limiting the potential to develop effective pharmacological therapy for AAA.METHODS:In this observational cross-sectional study, formalin-fixed, paraffin-embedded tissues from 11 AAA patients and 12 controls were analyzed by Xenium spatial transcriptomics. Cellular states and localization within tissue architecture were mapped to identify cellular neighborhoods and infer cell–cell communication.RESULTS:We generated a high-resolution spatial transcriptomics atlas of 581,664 cells in 26 clusters. AAAs showed a significant loss of contractile smooth muscle cells, expansion of pro-angiogenic endothelial subsets, and broad infiltration of immune cells. These inflammatory changes were accompanied by expansion of activated, universal, and CXCL12+ adventitial fibroblasts. Spatial transcriptomic analysis revealed fibroblast–immune colocalization and adventitial tertiary lymphoid organs. Inferred signaling pathway analysis identified increased interactions between CXCL12+ fibroblasts and CXCR4+ T and B cells in the adventitia of AAAs. Fibroblasts that expressed CXCL12 had significantly more immune cell neighbors than fibroblasts that did not, suggesting that they serve as stromal hubs for adaptive immune clustering. Genome-wide association analysis linked AAA heritability to fibroblasts, modulated smooth muscle cells, and foamy macrophages.CONCLUSION:Our novel high-resolution spatial transcriptomic atlas of human AAAs revealed coordinated pathogenic reprogramming of stromal and immune cells, defined by smooth muscle cell depletion, fibroblast activation, endothelial remodeling, and disproportionate expansion of immune cells. Through CXCR4 signaling, CXCL12+ fibroblasts serve as central organizers of immune niches, suggesting stromal–immune crosstalk as a therapeutic target in AAA.

Objective To investigate the expression of microRNA(miRNA, miR)- 1260b in non-small-cell lung carcinoma(NSCLC) and paracarcinoma tissues and the clinical significance. Methods Seventy-eight pairs of lung cancer and paracarcinoma tissues were collected from patients sufferred from NSCLC. MicroRNA microarray was used to screen the miRNA with different expression levels in lung cancer and paracarcinoma tissues. The real-time quantitative polymerase chain reaction(Real-time PCR) was performed to detect the expression of target miRNA with enlarged clinical samples. The clinical analysis was carried out to explore the relationship between miR-1260b and clinical features. Results The ΔCt values of miR-1260b expression in lung cancer and paracarcinoma tissues were 10.04±1.43 and 10.97±1.17, respectively. Compared to paracarcinoma tissues, 82.2% cases showed increased expression of miR- 1260b in lung cancer tissues(P< 0.05). Clinical analysis demonstrated that there was no significant correlation between the expression level of miR-1260b with sex, age, differentiation degree and TNM classification, respectively. Conclusion These results suggested that miR-1260b expression in lung cancer was higher than in paracarcinoma tissues, and may play important roles in the development and progression of lung cancer. Key words: MicroRNA-1260b; Non-small-cell lung carcinoma

The lung tumor microenvironment includes local infiltration of immune cells,mesenchymal cells,cytokines and lung cancer cells.The infiltration of immune cells in the lung tumor microenvironment is involved in lung cancer progression and immune escape.This article discusses infiltration characteristics,function,and relationship of these cells,explores the role of tumor microenvironment in process and development of lung cancer. Key words: Lung cancer; Tumor microenvironment ; Immune cells; Immune escape

e21025 Background: The tumor microenvironment (TME) plays an important role in tumor progression and treatment response, therefore profoundly affecting patient outcomes. Efforts to characterize the TME in lung adenocarcinoma are emerging but have been limited by the sample size and lack of treatment timepoints. Methods: We characterized changes in lung TME using the xCell algorithm to distinguish 64 immune and stroma cell types from bulk RNA sequencing data. The correlation between subtype cell population in lung TME and various clinical and biological characteristics was analyzed from over 500 lung adenocarcinoma (LUAD) samples from The Cancer Genome Atlas and an independent cohort of 48 advanced LUAD patients with treatment annotations (treatment-naive, residual disease, and progressive disease). In addition, we used key features in lung TME to predict prognosis using deep learning algorithms. Results: We found significant changes in both immune and stroma cell populations according to various clinical parameters such as smoking history, cancer stage, and treatment status. Specific sub-populations within lung TME correlate with survival outcomes based on Kaplan-Meier survival analyses. CD4- and CD8-positive T-cells are enriched in early stage disease and depleted in late stage disease, suggesting evolution of the TME during cancer progression. Consistent with previous reports, scores of immune cell populations associated with worse survival, such as T helper type 2 cells, are increased in late stage disease. Smoking history also reshapes the lung TME as populations correlated with better survival are decreased in smokers. We also found variations in sub-populations according to the driver oncogenes, with a less abundant lymphoid compartment in EGFR mutant samples compared to KRAS driven samples. Interestingly, we found higher scores of macrophage populations in residual disease following targeted-therapy treatment compared to pre-treatment. Finally, using machine and deep learning methods we identified a panel of 12 key features within the lung TME which could be used to predict prognosis. Conclusions: We comprehensively characterized immune and stroma cell type changes in the lung TME utilizing bulk RNA-seq data, and evaluated the association of sub-type cell populations with different clinical and biological features. Key features in lung TME could be used to predict prognosis.

Lung cancer is one of the leading causes of cancer death in the world. Human non-small cell lung carcinoma (NSCLC) accounts for almost 80 percent of lung cancer cases. Aberrant phosphoinositide 3-kinase (PI3K)/ Akt /mTOR signaling pathways play important roles and have been widely observed in the development of NSCLC. Previous studies indicated that garlic extracts such as diallyl disulfide (DADS) and diallyl trisulfide (DATS) could inhibit the proliferation of several types of cancer in vitro. However, the inhibitory effects of S-allylcysteine (SAC) on the growth of NSCLC have not been demonstrated yet. Therefore, we investigated whether consumption of SAC could prevent the growth of NSCLC in both in vitro and in vivo models. In the current study, SAC significantly inhibited the proliferation of human NSCLC A549 cells in vitro. Treatment of NF-κB inhibitor, Bay-11-7082, could significantly inhibit the proliferation of NSCLC A549 cells. Our results demonstrated that SAC significantly suppressed the activation of mTOR, NF-κB and cyclin D1 molecules in vitro. Furthermore, our results demonstrated that consumption of SAC significantly inhibited the growth of highly metastatic human NSCLC cells in tumor bearing mice. Bioluminescence imaging, pathological and immunohistochemical (IHC) staining results also indicated that SAC could effectively suppress the growth and malignant progression of human NSCLC in vivo. The chemopreventive effects of SAC were associated with suppression of mTOR and NF-κB molecules in vivo. These results suggested that SAC could act as an effective agent against the malignant progression of human non small cell lung cancer in both in vitro and in vivo models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4605. doi:10.1158/1538-7445.AM2011-4605

Loss of heterozygosity on chromosome 22q has been detected in approximately 60% of advanced nonsmall cell lung carcinoma (NSCLC) as well as small cell lung carcinoma (SCLC), suggesting the presence of a tumor suppressor gene on 22q that is involved in lung cancer progression. Here, we isolated a myosin family gene, MYO18B, located at chromosome 22q12.1 and found that it is frequently deleted, mutated, and hypermethylated in lung cancers. Somatic MYO18B mutations were detected in 19% (14/75) of lung cancer cell lines and 13% (6/46) of primary lung cancers of both SCLC and NSCLC types. MYO18B expression was reduced in 88% (30/34) of NSCLC and 47% (8/17) of SCLC cell lines. Its expression was restored by treatment with 5-aza-2'-deoxycytidine in 11 of 14 cell lines with reduced MYO18B expression, and the promoter CpG island of the MYO18B gene was methylated in 17% (8/47) of lung cancer cell lines and 35% (14/40) of primary lung cancers. Furthermore, restoration of MYO18B expression in lung carcinoma cells suppressed anchorage-independent growth. These results indicate that the MYO18B gene is a strong candidate for a novel tumor suppressor gene whose inactivation is involved in lung cancer progression.

Objective To discuss the expression patterns of hypoxia inducible factor(HIF)-1α, HIF- 2α and vascular endothelial growth factor(VEGF)in metastatic lung cancer tissues. Methods The immunohistochemical streptavid-in- peroxidase(SP) method was applied to detect the expression of HIF-1α, HIF- 2α and VEGF in 93 cases of surgicall resected metastatic lung cancer, and 57 cases of primary non- small- cell lung carcinoma(NSCLC)as blank control group. Results HIF-1α, HIF-2α and VEGF proteins were expressed in 72 cases of metastatic lung cancer and 58 cases of primary NSCLC with the positive rate being 77.4%, 62.3% and 74.2% respectively(P 0.05). Conclusion HIF-1α, HIF-2α and VEGF expression in lung metastases is increased, and HIF-1α expression was positively correlated with VEGF expression. Key words: Metastatic lung cancer; Hypoxia inducible factor-1α; Hypoxia inducible factor-2α; Vascular endothelial growth factor

BackgroundImmune cells within the tumor microenvironment (TME) play a vital role in regulating tumor progression. Therefore, immunotherapies that stimulate anti-tumor responses are of great interest for the treatment of various...