Molecular subtypes of lung adenocarcinoma present distinct immune tumor microenvironments.

Overcoming resistance to immune checkpoint inhibitors is an important issue in patients with non-small-cell lung cancer (NSCLC). Transcriptome analysis shows that adenocarcinoma can be divided into three molecular subtypes: terminal respiratory unit (TRU), proximal proliferative (PP), and proximal inflammatory (PI), and squamous cell carcinoma (LUSQ) into four. However, the immunological characteristics of these subtypes are not fully understood. In this study, we investigated the immune landscape of NSCLC tissues in molecular subtypes using a multi-omics dataset, including tumor-infiltrating leukocytes (TILs) analyzed using flow cytometry, RNA sequences, whole exome sequences, metabolomic analysis, and clinicopathologic findings. In the PI subtype, the number of TILs increased and the immune response in the tumor microenvironment (TME) was activated, as indicated by high levels of tertiary lymphoid structures, and high cytotoxic marker levels. Patient prognosis was worse in the PP subtype than in other adenocarcinoma subtypes. Glucose transporter 1 (GLUT1) expression levels were upregulated and lactate accumulated in the TME of the PP subtype. This could lead to the formation of an immunosuppressive TME, including the inactivation of antigen-presenting cells. The TRU subtype had low biological malignancy and "cold" tumor-immune phenotypes. Squamous cell carcinoma (LUSQ) did not show distinct immunological characteristics in its respective subtypes. Elucidation of the immune characteristics of molecular subtypes could lead to the development of personalized immune therapy for lung cancer. Immune checkpoint inhibitors could be an effective treatment for the PI subtype. Glycolysis is a potential target for converting an immunosuppressive TME into an antitumorigenic TME in the PP subtype.

Tumor-infiltrating Leukocyte Profiling Defines Three Immune Subtypes of NSCLC with Distinct Signaling Pathways and Genetic Alterations.

Resistance to immune checkpoint blockade remains challenging in patients with non-small cell lung cancer (NSCLC). Tumor-infiltrating leukocyte (TIL) quantity, composition, and activation status profoundly influence responsiveness to cancer immunotherapy. This study examined the immune landscape in the NSCLC tumor microenvironment by analyzing TIL profiles of 281 fresh resected NSCLC tissues. Unsupervised clustering based on numbers and percentages of 30 TIL types classified adenocarcinoma (LUAD) and squamous cell carcinoma (LUSQ) into the cold, myeloid cell-dominant, and CD8+ T cell-dominant subtypes. These were significantly correlated with patient prognosis; the myeloid cell subtype had worse outcomes than the others. Integrated genomic and transcriptomic analyses, including RNA sequencing, whole-exome sequencing, T-cell receptor repertoire, and metabolomics of tumor tissue, revealed that immune reaction-related signaling pathways were inactivated, while the glycolysis and K-ras signaling pathways activated in LUAD and LUSQ myeloid cell subtypes. Cases with ALK and ROS1 fusion genes were enriched in the LUAD myeloid subtype, and the frequency of TERT copy-number variations was higher in LUSQ myeloid subtype than in the others. These classifications of NSCLC based on TIL status may be useful for developing personalized immune therapies for NSCLC. Significance: The precise TIL profiling classified NSCLC into novel three immune subtypes that correlates with patient outcome, identifying subtype-specific molecular pathways and genomic alterations that should play important roles in constructing subtype-specific immune tumor microenvironments. These classifications of NSCLC based on TIL status are useful for developing personalized immune therapies for NSCLC.

Imatinib Mesylate Exerted Antitumor Effect by Promoting Infiltration of Effector T Cells in Tumor.

Imatinib mesylate is a potent tyrosine kinase inhibitor that may induce immunological effects, such as inhibition of immune suppressive cells; but, how it modulates the immune system remains to be completely elucidated. In this study, we showed that cell proliferation of CT26 colon cancer and Lewis lung carcinoma (3LL) lung cancer cells was not inhibited by imatinib in vitro, although its administration significantly suppressed the growth of CT26, but not 3LL, subcutaneous tumors, and prolonged survival in CT26 tumor-bearing mice. Further, we examined the expression of immune cell-related molecules in the tumors to elucidate the differences in imatinib-mediated antitumor effects between CT26 and 3LL tumors. The nCounter assay showed that the expression of CD8 and CD8+ T cell-recruiting chemokine genes was significantly elevated in imatinib-treated CT26 tumors than that in control tumors; however, the gene expression remained unchanged in imatinib-treated or control 3LL tumors. Furthermore, frequency of interferon-γ+ (IFN-γ+) CD8+ T cells was increased in imatinib-treated CT26 tumors than control tumors, indicating induction of antitumor immunity by imatinib. The analysis indicates that imatinib promotes infiltration of effector T cells in tumors by upregulating expression of cytokines that recruit CD8+ T cells in the tumor microenvironment, which may lead to a strong antitumor effect.

Stemness and immune evasion conferred by the TDO2-AHR pathway are associated with liver metastasis of colon cancer.

The aryl hydrocarbon receptor (AHR) pathway modulates the immune system in response to kynurenine, an endogenous tryptophan metabolite. IDO1 and TDO2 catalyze kynurenine production, which promotes cancer progression by compromising host immunosurveillance. However, it is unclear whether the AHR activation regulates the malignant traits of cancer such as metastatic capability or cancer stemness. Here, we carried out systematic analyses of metabolites in patient-derived colorectal cancer spheroids and identified high levels of kynurenine and TDO2 that were positively associated with liver metastasis. In a mouse colon cancer model, TDO2 expression substantially enhanced liver metastasis, induced AHR-mediated PD-L1 transactivation, and dampened immune responses; these changes were all abolished by PD-L1 knockout. In patient-derived cancer spheroids, TDO2 or AHR activity was required for not only the expression of PD-L1, but also for cancer stem cell (CSC)-related characteristics and Wnt signaling. TDO2 was coexpressed with both PD-L1 and nuclear ß-catenin in colon xenograft tumors, and the coexpression of TDO2 and PD-L1 was observed in clinical colon cancer specimens. Thus, our data indicate that the activation of the TDO2-kynurenine-AHR pathway facilitates liver metastasis of colon cancer via PD-L1-mediated immune evasion and maintenance of stemness.

Association between increased peripheral blood CD86-positive plasmacytoid dendritic cells and immune-related adverse events in patients with non-small cell lung cancer.

The occurrence of immune-related adverse events (irAEs) after immune checkpoint inhibitors (ICIs) is unpredictable. Profiles of peripheral blood mononuclear cells (PBMCs) represent the host immune system and have the potential to predict irAEs. We analyzed PBMC subsets using multicolor flow cytometry before and at weeks 2 and 8 after the start of ICIs in patients with non-small cell lung cancer. Sixteen eligible patients were evaluated. The irAEs occurred in 6 patients (37.5%): diarrhea in 2, diarrhea and a rash in 1, pituitary dysfunction in 1, cholangitis in 1, and pneumonitis in 1. Patients experiencing irAEs had higher levels of CD86+plasmacytoid dendritic cells (pDCs) at the baseline and weeks 2 and 8 after the ICIs than those not experiencing irAEs (p = 0.005, 0.038, and 0.050, respectively). In patients experiencing irAEs, the levels of CD86+pDCs significantly decreased at weeks 2 and 8 compared to the baseline (p = 0 .034 and 0.025, respectively) but did not change in those not experiencing irAEs. The levels of other PBMC subsets were not significantly associated with irAEs. Higher levels of natural killer (NK) cells were significantly associated with an overall objective response (p = 0.024). In conclusion, higher levels of CD86+pDCs at the baseline and a reduction in those levels 2 and 8 weeks after ICIs were associated with the occurrence of irAEs. Higher levels of NK cells were associated with an objective response to ICIs. Evaluation of PBMCs may help to predict the efficacy and safety of ICIs.

Feasibility study of nivolumab as neoadjuvant chemotherapy for locally esophageal carcinoma: FRONTiER (JCOG1804E).

One of the standard treatments of resectable esophageal squamous cell carcinoma (ESCC) is neoadjuvant chemotherapy followed by surgery. Nivolumab showed efficacy for metastatic ESCC. However, the safety and efficacy of neoadjuvant nivolumab with chemotherapy for ESCC is unknown. Therefore, we will conduct FRONTiER to evaluate the safety and efficacy of nivolumab adding to neoadjuvant chemotherapy. FRONTiER comprises four experimental cohorts: (A) including nivolumab plus 5-FU+CDDP (cisplatin and 5-fluorouracil [CF]); (B) including one prior administration of nivolumab and the cohort A regimen; (C) including nivolumab plus docetaxel+ CF; (D) including one prior administration of nivolumab and the cohort C regimen; an expanded cohort. The primary end point is the incidence of dose-limiting toxicities from the initial dose to the 30th postoperative day. Clinical Trial Identifier: NCT03914443.

Local Administration of GITR Agonistic Antibody Induces a Stronger Antitumor Immunity than Systemic Delivery.

An anti-glucocorticoid induced TNF receptor (GITR) agonistic antibody (Ab) induces an antitumor immunity with both stimulation of effector T cells and inhibition of regulatory T cell activity. To enhance GITR Ab-mediated tumor immunity, we focused on the intratumoral route, since a tumor-localized high concentration of Ab would confer activation of only tumor-infiltrating T cells. First, in a murine colon cancer model, we showed that the intratumoral delivery of Ab significantly increased the number of effector T cells infiltrated into tumors, and suppressed tumor growth more effectively than the intraperitoneal and intravenous injections did. Then, we found that the injection of Ab into the peritumoral area induced a systemic antitumor immunity at a similar level to the intratumoral injection. Therefore, we hypothesized that the transfer of locally administrated Ab into tumor-draining lymph nodes (TDLNs) plays an important role in inducing an effective immunity. In fact, intratumorally or peritumorally injected Ab was detected in TDLNs, and resection of Ab-injected TDLNs significantly reduced GITR Ab-mediated systemic tumor immunity. Intratumoral injection showed less number of auto-reactive T cells in the spleen than the intraperitoneal injection did. Intratumoral delivery of GITR Ab is a promising approach to induce an effective immunity compared to the systemic delivery.

Pre-immunization of donor lymphocytes with GITR agonistic antibody enhances antitumor immunity in autologous hematopoietic stem cell transplantation.

The lymphopenic condition following autologous hematopoietic stem cell transplantation (HSCT) enhances the proliferation of T cells by engaging tumor-associated antigens, leading to the alteration of the T-cell repertoire towards antitumor immunity. However, cure by autologous HSCT alone have rarely occurred in the clinical setting. Since tumor-reactive lymphocytes preferentially proliferate during reconstitution of the immune system, we examined whether the priming of donor lymphocytes can strengthen the antitumor effect by HSCT in a CT26 murine colon cancer model. The systemic administration of an anti-glucocorticoid-induced TNF receptor (GITR) agonistic antibody (Ab) significantly increased the number of CT26-responsive T cells but not that of auto-reactive lymphocytes in donor mice. The infusion of non-primed and GITR Ab-primed donor lymphocytes suppressed the CT26 tumor growth, and only the primed lymphocytes eliminated tumors in all the treated mice. The frequency of CT26-responsive T cells was elevated in recipient mice infused with both primed and non-primed lymphocytes until 4 weeks after transplantation, while the frequency in recipients with primed lymphocytes was markedly elevated compared with that in mice harboring non-primed lymphocytes at 2 weeks. The frequencies of regulatory T cells and myeloid-derived suppressor cells were elevated in recipient mice infused with primed and non-primed lymphocytes 2 weeks after transplantation, and returned to normal levels by week 4. The combination of autologous HSCT with pre-immunization of donor lymphocytes is a promising strategy to induce strong antitumor immunity.