M1hot tumor-associated macrophages boost tissue-resident memory T cells infiltration and survival in human lung cancer.

BACKGROUND: The role of tumor-associated macrophages (TAMs) in determining the outcome between the antitumor effects of the adaptive immune system and the tumor's anti-immunity stratagems, is controversial. Macrophages modulate their activities and phenotypes by integration of signals in the tumor microenvironment. Depending on how macrophages are activated, they may adopt so-called M1-like, antitumor or M2-like, protumor profiles. In many solid tumors, a dominance of M2-like macrophages is associated with poor outcomes but in some tumor types, strong M1-like profiles are linked to better outcomes. We aimed to investigate the interrelationship of these TAM populations to establish how they modulate the efficacy of the adaptive immune system in early lung cancer. METHODS: Macrophages from matched lung (non-tumor-associated macrophages (NTAMs)) and tumor samples (TAMs) from resected lung cancers were assessed by bulk and single-cell transcriptomic analysis. Protein expression of genes characteristic of M1-like (chemokine (C-X-C motif) ligand 9) or M2-like (matrix metallopeptidase 12) functions was confirmed by confocal microscopy. Immunohistochemistry related the distribution of TAM transcriptomic signatures to density of CD8+ tissue-resident memory T cells (TRM) in tumors and survival data from an independent cohort of 393 patients with lung cancer. RESULTS: TAMs have significantly different transcriptomic profiles from NTAMs with >1000 differentially expressed genes. TAMs displayed a strong M2-like signature with no significant variation between patients. However, single-cell RNA-sequencing supported by immuno-stained cells revealed that additionally, in 25% of patients the M2-like TAMs also co-expressed a strong/hot M1-like signature (M1hot). Importantly, there was a strong association between the density of M1hot TAMs and TRM cells in tumors that was in turn linked to better survival. Our data suggest a mechanism by which M1hot TAMs may recruit TRM cells via CXCL9 expression and sustain them by making available more of the essential fatty acids on which TRM depend. CONCLUSIONS: We showed that in early lung cancer, expression of M1-like and M2-like gene signatures are not mutually exclusive since the same TAMs can simultaneously display both gene-expression profiles. The presence of M1hot TAMs was associated with a strong TRM tumor-infiltrate and better outcomes. Thus, therapeutic approaches to re-program TAMs to an M1hot phenotype are likely to augment the adaptive antitumor responses.

Targeting the Myofibroblastic Cancer-Associated Fibroblast Phenotype Through Inhibition of NOX4.

Background: Cancer-associated fibroblasts (CAFs) are tumor-promoting and correlate with poor survival in many cancers, which has led to their emergence as potential therapeutic targets. However, effective methods to manipulate these cells clinically have yet to be developed. Methods: CAF accumulation and prognostic significance in head and neck cancer (oral, n = 260; oropharyngeal, n = 271), and colorectal cancer (n = 56) was analyzed using immunohistochemistry. Mechanisms regulating fibroblast-to-myofibroblast transdifferentiation were investigated in vitro using RNA interference/pharmacological inhibitors followed by polymerase chain reaction (PCR), immunoblotting, immunofluorescence, and functional assays. RNA sequencing/bioinformatics and immunohistochemistry were used to analyze NAD(P)H Oxidase-4 (NOX4) expression in different human tumors. NOX4's role in CAF-mediated tumor progression was assessed in vitro, using CAFs from multiple tissues in Transwell and organotypic culture assays, and in vivo, using xenograft (n = 9-15 per group) and isograft (n = 6 per group) tumor models. All statistical tests were two-sided. Results: Patients with moderate/high levels of myofibroblastic-CAF had a statistically significant decrease in cancer-specific survival rates in each cancer type analyzed (hazard ratios [HRs] = 1.69-7.25, 95% confidence intervals [CIs] = 1.11 to 31.30, log-rank P ≤ .01). Fibroblast-to-myofibroblast transdifferentiation was dependent on a delayed phase of intracellular reactive oxygen species, generated by NOX4, across different anatomical sites and differentiation stimuli. A statistically significant upregulation of NOX4 expression was found in multiple human cancers (P < .001), strongly correlating with myofibroblastic-CAFs (r = 0.65-0.91, adjusted P < .001). Genetic/pharmacological inhibition of NOX4 was found to revert the myofibroblastic-CAF phenotype ex vivo (54.3% decrease in α-smooth muscle actin [α-SMA], 95% CI = 10.6% to 80.9%, P = .009), prevent myofibroblastic-CAF accumulation in vivo (53.2%-79.0% decrease in α-SMA across different models, P ≤ .02) and slow tumor growth (30.6%-64.0% decrease across different models, P ≤ .04). Conclusions: These data suggest that pharmacological inhibition of NOX4 may have broad applicability for stromal targeting across cancer types.

Evaluating the effect of immune cells on the outcome of patients with mesothelioma.

BACKGROUND: We systematically assessed the prognostic and predictive value of infiltrating adaptive and innate immune cells in a large cohort of patients with advanced mesothelioma. METHODS: A tissue microarray from 302 samples was constructed. Markers of adaptive immune response in T-cells (CD8+, FOXP3+, CD4+, CD45RO+, CD3+) and B-cells (CD20+), and of innate immune response; neutrophils (NP57+), natural killer cells (CD56+) and macrophages (CD68+) were evaluated. RESULTS: We found that in the epithelioid tumours, high CD4+ and CD20+ counts, and low FOXP3+, CD68+ and NP57+ counts linked to better outcome. In the non-epithelioid group low CD8+ and low FOXP3+ counts were beneficial.On multivariate analysis low FOXP3+ remained independently associated with survival in both groups. In the epithelioid group additionally high CD4+, high CD20+, and low NP57+ counts were prognostic. CONCLUSIONS: Our data demonstrate for the first time, in predominately advanced disease, the association of key markers of adaptive and innate immunity with survival and the differential effect of histology. A better understanding of the immunological drivers of the different subtypes of mesothelioma will assist prognostication and disease-specific clinical decision-making.

Pro-migratory and TGF-ß-activating functions of αvß6 integrin in pancreatic cancer are differentially regulated via an Eps8-dependent GTPase switch.

The integrin αvß6 is up-regulated in numerous carcinomas, where expression commonly correlates with poor prognosis. αvß6 promotes tumour invasion, partly through regulation of proteases and cell migration, and is also the principal mechanism by which epithelial cells activate TGF-ß1; this latter function complicates therapeutic targeting of αvß6, since TGF-ß1 has both tumour-promoting and -suppressive effects. It is unclear how these different αvß6 functions are linked; both require actin cytoskeletal reorganization, and it is suggested that tractive forces generated during cell migration activate TGF-ß1 by exerting mechanical tension on the ECM-bound latent complex. We examined the functional relationship between cell invasion and TGF-ß1 activation in pancreatic ductal adenocarcinoma (PDAC) cells, and confirmed that both processes are αvß6-dependent. Surprisingly, we found that cellular functions could be biased towards either motility or TGF-ß1 activation depending on the presence or absence of epidermal growth factor receptor pathway substrate 8 (Eps8), a regulator of actin remodelling, endocytosis, and GTPase activation. Similar to αvß6, we found that Eps8 was up-regulated in >70% of PDACs. In complex with Abi1/Sos1, Eps8 regulated αvß6-dependent cell migration through activation of Rac1. Down-regulation of Eps8, Sos1 or Rac1 suppressed cell movement, while simultaneously increasing αvß6-dependent TGF-ß1 activation. This latter effect was modulated through increased cell tension, regulated by Rho activation. Thus, the Eps8/Abi1/Sos1 tricomplex acts as a key molecular switch altering the balance between Rac1 and Rho activation; its presence or absence in PDAC cells modulates αvß6-dependent functions, resulting in a pro-migratory (Rac1-dependent) or a pro-TGF-ß1 activation (Rho-dependent) functional phenotype, respectively. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Mycobacterium tuberculosis subverts negative regulatory pathways in human macrophages to drive immunopathology.

Tuberculosis remains a global pandemic and drives lung matrix destruction to transmit. Whilst pathways driving inflammatory responses in macrophages have been relatively well described, negative regulatory pathways are less well defined. We hypothesised that Mycobacterium tuberculosis (Mtb) specifically targets negative regulatory pathways to augment immunopathology. Inhibition of signalling through the PI3K/AKT/mTORC1 pathway increased matrix metalloproteinase-1 (MMP-1) gene expression and secretion, a collagenase central to TB pathogenesis, and multiple pro-inflammatory cytokines. In patients with confirmed pulmonary TB, PI3Kδ expression was absent within granulomas. Furthermore, Mtb infection suppressed PI3Kδ gene expression in macrophages. Interestingly, inhibition of the MNK pathway, downstream of pro-inflammatory p38 and ERK MAPKs, also increased MMP-1 secretion, whilst suppressing secretion of TH1 cytokines. Cross-talk between the PI3K and MNK pathways was demonstrated at the level of eIF4E phosphorylation. Mtb globally suppressed the MMP-inhibitory pathways in macrophages, reducing levels of mRNAs encoding PI3Kδ, mTORC-1 and MNK-1 via upregulation of miRNAs. Therefore, Mtb disrupts negative regulatory pathways at multiple levels in macrophages to drive a tissue-destructive phenotype that facilitates transmission.

Tissue-resident memory features are linked to the magnitude of cytotoxic T cell responses in human lung cancer.

Therapies that boost the anti-tumor responses of cytotoxic T lymphocytes (CTLs) have shown promise; however, clinical responses to the immunotherapeutic agents currently available vary considerably, and the molecular basis of this is unclear. We performed transcriptomic profiling of tumor-infiltrating CTLs from treatment-naive patients with lung cancer to define the molecular features associated with the robustness of anti-tumor immune responses. We observed considerable heterogeneity in the expression of molecules associated with activation of the T cell antigen receptor (TCR) and of immunological-checkpoint molecules such as 4-1BB, PD-1 and TIM-3. Tumors with a high density of CTLs showed enrichment for transcripts linked to tissue-resident memory cells (TRM cells), such as CD103, and CTLs from CD103hi tumors displayed features of enhanced cytotoxicity. A greater density of TRM cells in tumors was predictive of a better survival outcome in lung cancer, and this effect was independent of that conferred by CTL density. Here we define the 'molecular fingerprint' of tumor-infiltrating CTLs and identify potentially new targets for immunotherapy.

Induction of fibroblast senescence generates a non-fibrogenic myofibroblast phenotype that differentially impacts on cancer prognosis.

Cancer-associated fibroblasts (CAF) remain a poorly characterized, heterogeneous cell population. Here we characterized two previously described tumor-promoting CAF sub-types, smooth muscle actin (SMA)-positive myofibroblasts and senescent fibroblasts, identifying a novel link between the two. Analysis of CAF cultured ex vivo, showed that senescent CAF are predominantly SMA-positive; this was confirmed by immunochemistry in head & neck (HNSCC) and esophageal (EAC) cancers. In vitro, we found that fibroblasts induced to senesce develop molecular, ultrastructural and contractile features typical of myofibroblasts and this is dependent on canonical TGF-ß signaling. Similar to TGF-ß1-generated myofibroblasts, these cells secrete soluble factors that promote tumor cell motility. However, RNA-sequencing revealed significant transcriptomic differences between the two SMA-positive CAF groups, particularly in genes associated with extracellular matrix (ECM) deposition and organization, which differentially promote tumor cell invasion. Notably, second harmonic generation imaging and bioinformatic analysis of SMA-positive human HNSCC and EAC showed that collagen fiber organization correlates with poor prognosis, indicating that heterogeneity within the SMA-positive CAF population differentially impacts on survival. These results show that non-fibrogenic, SMA-positive myofibroblasts can be directly generated through induction of fibroblast senescence and suggest that senescence and myofibroblast differentiation are closely linked processes.

Tumour infiltrating lymphocytes correlate with improved survival in patients with oesophageal adenocarcinoma.

BACKGROUND: Oesophageal adenocarcinoma (OAC) is increasingly common in the west, and survival remains poor at 10-15 % at 5 years. Immune responses are increasingly implicated as a determining factor of tumour progression. The ability of lymphocytes to recognise tumour antigens provides a mechanism for a host immune attack against cancer providing a potential treatment strategy. MATERIALS AND METHODS: Tumour infiltrating lymphocytes (TILs: CD3+, CD4+, CD8+ and FOXp3+) were assessed by immunohistochemistry using tissue microarrays in a contemporary and homogeneous cohort of OAC patients (n = 128) undergoing curative treatment. RESULTS: Multivariate analysis identified three independent prognostic factors for improved cancer-specific survival (CSS): increased CD8+ TILs (p = 0.003), completeness of resection (p < 0.0001) and lower pathological N stage (p < 0.0001). Independent prognostic factors for favourable disease-free survival included surgery-only treatment (p = 0.015), completeness of resection (p = 0.001), increased CD8+ TILs (p < 0.0001) and reduced pathological N stage (p < 0.0001). Higher levels of TILs in the pathological specimen were associated with significant pathological response to neoadjuvant chemotherapy (NAC). On multivariate analysis increased levels of CD4+ (p = 0.017) and CD8+ TILs (p = 0.005) were associated with significant local tumour regression and lymph node downstaging, respectively. DISCUSSION: Our results establish an association of TILs and survival in OAC, as seen in other solid tumours, and identify particular TIL subsets that are present at higher levels in patients who responded to NAC compared to non-responders. These findings highlight potential therapeutic strategies in EAC based on utilising the host immunological response and highlight the immune responses biomarker potential.

A subset of myofibroblastic cancer-associated fibroblasts regulate collagen fiber elongation, which is prognostic in multiple cancers.

Collagen structure has been shown to influence tumor cell invasion, metastasis and clinical outcome in breast cancer. However, it remains unclear how it affects other solid cancers. Here we utilized multi-photon laser scanning microscopy and Second Harmonic Generation to identify alterations to collagen fiber structure within the tumor stroma of head & neck, esophageal and colorectal cancers. Image segmentation algorithms were then applied to quantitatively characterize these morphological changes, showing that elongated collagen fibers significantly correlated with poor clinical outcome (Log Rank p < 0.05). We used TGF-ß treatment to model fibroblast conversion to smooth muscle actin SMA-positive cancer associated fibroblasts (CAFs) and found that these cells induce the formation of elongated collagen fibers in vivo. However, proteomic/transcriptomic analysis of SMA-positive CAFs cultured ex-vivo showed significant heterogeneity in the expression of genes with collagen fibril organizing gene ontology. Notably, stratifying patients according to stromal SMA-positivity and collagen fiber elongation was found to provide a highly significant correlation with poor survival in all 3 cancer types (Log Rank p ≤ 0.003). In summary, we show that increased collagen fiber length correlates with poor patient survival in multiple tumor types and that only a sub-set of SMA-positive CAFs can mediate the formation of this collagen structure.

Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival.

Acidification of the extracellular and/or intracellular environment is involved in many aspects of cell physiology and pathology. Eukaryotic elongation factor 2 kinase (eEF2K) is a Ca(2+)/calmodulin-dependent kinase that regulates translation elongation by phosphorylating and inhibiting eEF2. Here we show that extracellular acidosis elicits activation of eEF2K in vivo, leading to enhanced phosphorylation of eEF2. We identify five histidine residues in eEF2K that are crucial for the activation of eEF2K during acidosis. Three of them (H80, H87, and H94) are in its calmodulin-binding site, and their protonation appears to enhance the ability of calmodulin to activate eEF2K. The other two histidines (H227 and H230) lie in the catalytic domain of eEF2K. We also identify His108 in calmodulin as essential for activation of eEF2K. Acidification of cancer cell microenvironments is a hallmark of malignant solid tumors. Knocking down eEF2K in cancer cells attenuated the decrease in global protein synthesis when cells were cultured at acidic pH. Importantly, activation of eEF2K is linked to cancer cell survival under acidic conditions. Inhibition of eEF2K promotes cancer cell death under acidosis.

Staging and treatment of oropharyngeal cancer in the human papillomavirus era.

BACKGROUND: Oropharyngeal squamous cell carcinoma (SCC) is staged using the TNM system. Human papillomavirus (HPV)-positive tumors have improved prognosis, despite presenting at advanced stage. Optimal treatment and stratification of HPV-positive patients are not clearly defined. METHODS: We retrospectively analyzed 266 patients with oropharyngeal SCC for mortality and feeding tube dependency related to TNM stage, HPV status, and treatment. RESULTS: TNM staging was prognostic in HPV-negative patients (stage III/IV hazard ratio [HR], 2.00; p = .05; N(+) HR, 2.19; p = .02). Only T classification was prognostic in HPV-positive tumors (T3/T4 HR 3.31; p = .006). HPV-positive tumors showed improved survival regardless of treatment. Patients receiving chemotherapy had a significantly increased risk of feeding tube dependency (odds ratio [OR], 1.72; p = .03). CONCLUSION: These data suggest that the current TNM system has little prognostic value in HPV-positive oropharyngeal SCC. Patients with HPV-positive tumors show improved survival independent of treatment. The addition of chemotherapy increases the risk of feeding tube dependency and could potentially be avoided in T1/T2 HPV-positive tumors without compromising survival.

Suppression of Hedgehog signalling promotes pro-tumourigenic integrin expression and function.

Aberrant Hedgehog (Hh) signalling has been reported in a number of malignancies, particularly basal cell carcinoma (BCC) of the skin. Clinical trials of Hh inhibitors are underway in many cancers, and these have produced significant clinical benefit in BCC patients, although regrowth of new, or clinically aggressive, variants, as well as development of secondary malignancies, has been reported. αvß6 integrin is expressed in many cancers, where it has been shown to correlate with an aggressive tumour phenotype and poor prognosis. We have previously reported αvß6 up-regulation in aggressive, morphoeic BCC variants, where it modulates the stromal response and induces invasion. To examine a possible link between Hh and αvß6 function, we generated BCC models, overexpressing Gli1 in immortalized keratinocytes (NTert1, HaCaT). Unexpectedly, we found that suppressing Gli1 significantly increased αvß6 expression. This promoted tumour cell motility and also stromal myofibroblast differentiation through integrin-dependent TGF-ß1 activation. Gli1 inhibited αvß6 expression by suppressing TGF-ß1-induced Smad2/3 activation, blocking a positive feedback loop maintaining high αvß6 levels. A similar mechanism was observed in AsPC1 pancreatic cancer cells expressing endogenous Gli1, suggesting a common mechanism across tumour types. In vitro findings were supported using human clinical samples, where we showed an inverse correlation between αvß6 and Gli1 expression in different BCC subtypes and pancreatic cancers. In summary, we show that expression of Gli1 and αvß6 inversely correlates in tumours in vivo, and Hh targeting up-regulates TGF-ß1/Smad2/3-dependent αvß6 expression, promoting pro-tumourigenic cell functions in vitro. These results have potential clinical significance, given the reported recurrence of BCC variants and secondary malignancies in patients treated by Hh targeting.