Author’s Accepted Manuscript Analysis of non-small cell lung cancer microenvironment indicates preponderance of T cell exhaustion marker expression Hui Zhou, Tingwei Liu, Zanfeng Wang www.elsevier.com/locate/yexcr
PII: DOI: Reference:
S0014-4827(17)30490-1 http://dx.doi.org/10.1016/j.yexcr.2017.09.008 YEXCR10730
To appear in: Experimental Cell Research Received date: 24 August 2017 Revised date: 4 September 2017 Accepted date: 6 September 2017 Cite this article as: Hui Zhou, Tingwei Liu and Zanfeng Wang, Analysis of nonsmall cell lung cancer microenvironment indicates preponderance of T cell exhaustion marker expression, Experimental Cell Research, http://dx.doi.org/10.1016/j.yexcr.2017.09.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Analysis of non-small cell lung cancer microenvironment indicates preponderance of T cell exhaustion marker expression Hui Zhou, Tingwei Liu, Zanfeng Wang* Department of Respiratory Medicine, The First Affiliated Hospital of China Medical University, Shenyang, 110001, Liaoning, China *Correspondence to: Department of Respiratory Medicine, First Affiliated Hospital of China Medical University, No. 155 Nanjing North Street, Heping District, Shenyang, Liaoning 110001, China. Tel./fax: +86-024-83282530. [email protected]
Abstract Lung cancer metastasis causes 70% of an estimated 1.4 million deaths per annum. The major shortcoming in lung cancer is the tendency to have inherent or develop acquired resistance to chemotherapy. It is now evolving that such resistance might develop due to differential contribution and interaction with tumor microenvironment, stromal cells, and the extracellular matrix. The objective of the current study was to define the lung cancer tumor microenvironment. We have identified multiple tumor-infiltrating T lymphocyte subsets in patients with lung cancer, which were independent of disease stage. Functional analysis indicated high expression of the inhibitory receptors, cytotoxic T-lymphocyte-associated protein 4 (CTLA4), lymphocyte activated gene 3 (LAG3) and programmed cell death protein 1 (PD-1) in both CD4 and CD8 subsets, compared to non-malignant controls. Inhibitory receptors expressed by the tumor infiltrating T cells might mediate tolerance to tumor antigens with co-expression of these receptors exacerbating lung carcinogenesis and metastatic progression.
1
Abbreviations CTLA4: cytotoxic T-lymphocyte-associated protein 4; LAG3: lymphocyte activated gene 3; PD1: programmed cell death protein 1; CFSE: carboxyfluorescein succinimidyl ester; CD25 neg: CD25Dep cells
Keywords：lung cancer; microenvironment; T cell; exhaustion marker 1. Introduction With an estimated 1.4 million deaths per annum, lung cancer is a leading cause of cancer mortality [1, 2]. Metastatic spread accounts for more than 70% of these deaths [3]. Hence it is imperative to increase our understanding of the pathogenic mechanisms underlying lung cancer origin and disease progression. One factor that complicates outcome in lung cancer patients is acquired resistance to chemotherapy [4]. Recently, the contribution of the tumor microenvironment and extracellular matrix to lung cancer progression to resistance to chemotherapy has started to be investigated [5]. Cancer metastasis is an unusually inefficient process with only a few cells from the primary tumor surviving the interactions with the extracellular matrix and microenvironment and reaching the secondary site. Hence, discovering the players and mechanisms by which tumor cells interplay with the tumor microenvironment will uncover key therapeutic targets. Tumor-host immune interaction affects tumor development, progression, prognosis [6]. It is likely some of the pathologic manifestations in lung cancer are driven by interactions with normal immune cells reacting to the pathologic cells. In this context, and even if our current understanding of the tumor immune microenvironment in lung cancer remains limited when
2
compared with other cancers, several data suggest that exploring the immune system is an interesting strategy. Characterizing the population, function, and interactions of the lung cancer lesion associated immune cells may provide novel therapeutic opportunities. Hence, the objective of the current study was to define contribution of the microenvironment in lung cancer disease pathogenesis. Flow cytometric analysis of tumor infiltrating CD3+ T cells in lung cancer revealed high expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA4), lymphocyte activated gene 3 (LAG3) and programmed cell death protein 1 (PD-1) compared to non-malignant controls, indicating that effector T cell exhaustion might be an important mechanism of immune dysregulation in lung cancer patients.
2. Materials and Methods 2.1 Study patients and samples Biopsy tissue and blood samples were collected from lung patients under a protocol approved by the Institutional Review Board of The First Affiliated Hospital of China Medical University. Clinical data was recorded for each patient. Samples used in flow cytometry were solid tissue cells from 30 patient biopsies as well as tumor adjacent normal tissue for controls. Samples were processed in the same manner in accordance to protocols approved by the IRB and were frozen down prior to acquisition for cytometry. Some samples were stimulated for 18 hours prior to acquisition for flow cytometry but majority were stained immediately upon thawing. 2.2 Flow Cytometry The phenotype of lung cancer tumor microenvironment cells was evaluated by conventional dual color immunofluorescence using indicated fluorescence-conjugated antibodies (Becton–
3
Dickinson, MountainView, CA, USA) as per standard protocols using flow cytometry (BD FACS CaliburTM, Mississauga, ON, Canada). The data were analyzed by FlowJo software (TreeStar Inc., Ashland, OR, USA). Live cells were identified by forward and side scatter. Gating was based upon isotype controls. The percentage of positive cells was calculated by subtracting the value of the isotype control. The absolute numbers of positive cells per ml were calculated as percentage of positive cells.
2.3 Suppression assays The in vitro suppressive capacity of freshly isolated nTregs, or Tregs isolated from LCH lesion or control tonsils, and CD25-depleted cells was assessed using a carboxyfluorescein succinimidyl ester (CFSE)-based inhibition assay [7]. PBMC were labeled with 1.5 M CFSE (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions, and then stimulated with irradiated (40 Gy) allogeneic PBMC (at a 4:1 effector/feeders ratio) and OKT3 (0.5 mg/mL). To assess their suppressive capacity, Tregs and expanded CD8+ cells were added to the culture at a 1:1 ratio. On Day 7 of culture, cells were labeled with PE-, PerCP, or APCconjugated CD4 and CD8 monoclonal antibodies and CFSE dilution was analyzed using a FACSCalibur to measure cell proliferation. Suppression was expressed as the percentage of inhibition of T-cell proliferation in the presence of Tregs as compared to control cells.
2.4 Statistical analysis All in vitro experiments were summarized as mean ± SD, unless otherwise indicated. Student’s ttest was used to determine the statistical significant differences between samples, with P < 0.05 indicating a statistically significant difference.
4
3. Results 3.1 Characterization of immune microenvironment in lung cancer patients To identify the different cells that form the lung cancer lesion, we used flow cytometry to simultaneously measure the content of multiple markers in cells obtained from biopsies from 30 patients with lung cancer ranging from Stage I to Stage IV. The results were compared to expression of the same markers in tumor adjacent normal lung tissue.
CD3+ T cells were the most prominent population in cancerous lung lesions (12.5% ±10% in malignant versus 4% ± 7% in non-malignant lung; P0.05), and CD68+ macrophages (0.94% ± 0.82% in LCH versus 2.3% ± 2.8% in healthy skin; P>0.05) were not appreciably present in the lung tumor infiltrate, and did not appreciably differ between malignant lesion and tumor adjacent normal lung tissue (Fig. 1C). We next analyzed the CD3+ T cell subset (Fig. 2A). Whereas, in 15/25 cases, helper CD3+CD4+ T cells were significantly higher than effector CD3+CD8+ T cells (62% ± 20% CD4 versus 35% ± 10% CD8; P0.05) (Fig. 2A). The number of naïve T cells within the CD4+ (1.97% ± 1.3% in malignant versus 24.3% ± 7.1% in non-malignant; P
PII: DOI: Reference:
S0014-4827(17)30490-1 http://dx.doi.org/10.1016/j.yexcr.2017.09.008 YEXCR10730
To appear in: Experimental Cell Research Received date: 24 August 2017 Revised date: 4 September 2017 Accepted date: 6 September 2017 Cite this article as: Hui Zhou, Tingwei Liu and Zanfeng Wang, Analysis of nonsmall cell lung cancer microenvironment indicates preponderance of T cell exhaustion marker expression, Experimental Cell Research, http://dx.doi.org/10.1016/j.yexcr.2017.09.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Analysis of non-small cell lung cancer microenvironment indicates preponderance of T cell exhaustion marker expression Hui Zhou, Tingwei Liu, Zanfeng Wang* Department of Respiratory Medicine, The First Affiliated Hospital of China Medical University, Shenyang, 110001, Liaoning, China *Correspondence to: Department of Respiratory Medicine, First Affiliated Hospital of China Medical University, No. 155 Nanjing North Street, Heping District, Shenyang, Liaoning 110001, China. Tel./fax: +86-024-83282530. [email protected]
Abstract Lung cancer metastasis causes 70% of an estimated 1.4 million deaths per annum. The major shortcoming in lung cancer is the tendency to have inherent or develop acquired resistance to chemotherapy. It is now evolving that such resistance might develop due to differential contribution and interaction with tumor microenvironment, stromal cells, and the extracellular matrix. The objective of the current study was to define the lung cancer tumor microenvironment. We have identified multiple tumor-infiltrating T lymphocyte subsets in patients with lung cancer, which were independent of disease stage. Functional analysis indicated high expression of the inhibitory receptors, cytotoxic T-lymphocyte-associated protein 4 (CTLA4), lymphocyte activated gene 3 (LAG3) and programmed cell death protein 1 (PD-1) in both CD4 and CD8 subsets, compared to non-malignant controls. Inhibitory receptors expressed by the tumor infiltrating T cells might mediate tolerance to tumor antigens with co-expression of these receptors exacerbating lung carcinogenesis and metastatic progression.
1
Abbreviations CTLA4: cytotoxic T-lymphocyte-associated protein 4; LAG3: lymphocyte activated gene 3; PD1: programmed cell death protein 1; CFSE: carboxyfluorescein succinimidyl ester; CD25 neg: CD25Dep cells
Keywords：lung cancer; microenvironment; T cell; exhaustion marker 1. Introduction With an estimated 1.4 million deaths per annum, lung cancer is a leading cause of cancer mortality [1, 2]. Metastatic spread accounts for more than 70% of these deaths [3]. Hence it is imperative to increase our understanding of the pathogenic mechanisms underlying lung cancer origin and disease progression. One factor that complicates outcome in lung cancer patients is acquired resistance to chemotherapy [4]. Recently, the contribution of the tumor microenvironment and extracellular matrix to lung cancer progression to resistance to chemotherapy has started to be investigated [5]. Cancer metastasis is an unusually inefficient process with only a few cells from the primary tumor surviving the interactions with the extracellular matrix and microenvironment and reaching the secondary site. Hence, discovering the players and mechanisms by which tumor cells interplay with the tumor microenvironment will uncover key therapeutic targets. Tumor-host immune interaction affects tumor development, progression, prognosis [6]. It is likely some of the pathologic manifestations in lung cancer are driven by interactions with normal immune cells reacting to the pathologic cells. In this context, and even if our current understanding of the tumor immune microenvironment in lung cancer remains limited when
2
compared with other cancers, several data suggest that exploring the immune system is an interesting strategy. Characterizing the population, function, and interactions of the lung cancer lesion associated immune cells may provide novel therapeutic opportunities. Hence, the objective of the current study was to define contribution of the microenvironment in lung cancer disease pathogenesis. Flow cytometric analysis of tumor infiltrating CD3+ T cells in lung cancer revealed high expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA4), lymphocyte activated gene 3 (LAG3) and programmed cell death protein 1 (PD-1) compared to non-malignant controls, indicating that effector T cell exhaustion might be an important mechanism of immune dysregulation in lung cancer patients.
2. Materials and Methods 2.1 Study patients and samples Biopsy tissue and blood samples were collected from lung patients under a protocol approved by the Institutional Review Board of The First Affiliated Hospital of China Medical University. Clinical data was recorded for each patient. Samples used in flow cytometry were solid tissue cells from 30 patient biopsies as well as tumor adjacent normal tissue for controls. Samples were processed in the same manner in accordance to protocols approved by the IRB and were frozen down prior to acquisition for cytometry. Some samples were stimulated for 18 hours prior to acquisition for flow cytometry but majority were stained immediately upon thawing. 2.2 Flow Cytometry The phenotype of lung cancer tumor microenvironment cells was evaluated by conventional dual color immunofluorescence using indicated fluorescence-conjugated antibodies (Becton–
3
Dickinson, MountainView, CA, USA) as per standard protocols using flow cytometry (BD FACS CaliburTM, Mississauga, ON, Canada). The data were analyzed by FlowJo software (TreeStar Inc., Ashland, OR, USA). Live cells were identified by forward and side scatter. Gating was based upon isotype controls. The percentage of positive cells was calculated by subtracting the value of the isotype control. The absolute numbers of positive cells per ml were calculated as percentage of positive cells.
2.3 Suppression assays The in vitro suppressive capacity of freshly isolated nTregs, or Tregs isolated from LCH lesion or control tonsils, and CD25-depleted cells was assessed using a carboxyfluorescein succinimidyl ester (CFSE)-based inhibition assay [7]. PBMC were labeled with 1.5 M CFSE (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions, and then stimulated with irradiated (40 Gy) allogeneic PBMC (at a 4:1 effector/feeders ratio) and OKT3 (0.5 mg/mL). To assess their suppressive capacity, Tregs and expanded CD8+ cells were added to the culture at a 1:1 ratio. On Day 7 of culture, cells were labeled with PE-, PerCP, or APCconjugated CD4 and CD8 monoclonal antibodies and CFSE dilution was analyzed using a FACSCalibur to measure cell proliferation. Suppression was expressed as the percentage of inhibition of T-cell proliferation in the presence of Tregs as compared to control cells.
2.4 Statistical analysis All in vitro experiments were summarized as mean ± SD, unless otherwise indicated. Student’s ttest was used to determine the statistical significant differences between samples, with P < 0.05 indicating a statistically significant difference.
4
3. Results 3.1 Characterization of immune microenvironment in lung cancer patients To identify the different cells that form the lung cancer lesion, we used flow cytometry to simultaneously measure the content of multiple markers in cells obtained from biopsies from 30 patients with lung cancer ranging from Stage I to Stage IV. The results were compared to expression of the same markers in tumor adjacent normal lung tissue.
CD3+ T cells were the most prominent population in cancerous lung lesions (12.5% ±10% in malignant versus 4% ± 7% in non-malignant lung; P0.05), and CD68+ macrophages (0.94% ± 0.82% in LCH versus 2.3% ± 2.8% in healthy skin; P>0.05) were not appreciably present in the lung tumor infiltrate, and did not appreciably differ between malignant lesion and tumor adjacent normal lung tissue (Fig. 1C). We next analyzed the CD3+ T cell subset (Fig. 2A). Whereas, in 15/25 cases, helper CD3+CD4+ T cells were significantly higher than effector CD3+CD8+ T cells (62% ± 20% CD4 versus 35% ± 10% CD8; P0.05) (Fig. 2A). The number of naïve T cells within the CD4+ (1.97% ± 1.3% in malignant versus 24.3% ± 7.1% in non-malignant; P
