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.

Fibroblast inhibition by tocilizumab enabled gemcitabine/nab-paclitaxel rechallenge for pancreatic cancer.

Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) pathway inhibition may overcome chemoresistance of metastatic pancreatic cancer (MPC). We sought to determine the safety and recommended dose of tocilizumab (TCZ), an IL-6 receptor monoclonal antibody, and biological correlates of tumor shrinkage in patients with gemcitabine (GEM)/nanoparticle albumin-bound paclitaxel (nab-PTX)-refractory MPC. This phase 1 study enrolled 10 patients with MPC who had progressed after GEM/nab-PTX. Patients initially received TCZ 8 mg/kg on Day 1 and nab-PTX 100 mg/m2 + GEM 750 mg/m2 on Days 2, 9, and 16. Before and at the end of Cycle 1, biopsy of liver metastases was performed 3-5 h after levofloxacin (LVFX) administration to measure LVFX infiltration into tumor tissue. No dose-limited toxicities occurred, and the recommended dosage of TCZ was determined to be 8 mg/kg. Treatment-emergent adverse events occurred in 80% of patients, of which decreased neutrophil count was the most common. Tumor reduction during Cycle 1 was observed in four patients, who were defined as responders. In paired-biopsy samples, responder-related biological activities were an increase of cleaved PARP expression of tumor nuclei (p = 0.01), a decrease of proliferative cancer-associated fibroblasts (CAFs) (p = 0.08), and an increase of LVFX infiltration in the tumor (p = 0.04). A decrease of phosphorylated STAT3 expression (p = 0.02) favored an increase in LVFX infiltration. In conclusion, TCZ + GEM/nab-PTX-rechallenge had a manageable safety profile and showed preliminary activity via inhibition of CAF and improved intratumoral drug infiltration in MPC.

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.

Protruding structures with high expression of LGR5 are formed during regrowth phase after chemo-treatment in xenograft model of colorectal adenocarcinoma.

Recurrence after chemotherapy is one of the biggest obstacles in cancer therapy, along with metastasis. Although histopathological evaluation in preclinical models like the xenograft model help us to understand the pathophysiological process of tumor growth, there are not enough detailed histopathological analyses of such models. In this study, to learn how a tumor recovers the typical tumor structure after structural corruption during chemo-treatment, xenografted tumors originating from a patient-derived xenograft of colorectal cancer (CRC) were analyzed histopathologically over time. There were many Duct (Flattened) at Day 1 (one day after the final administration of Irinotecan), but the ratio of Duct (Columnar) and Cribriform-structures typically found in colorectal adenocarcinoma-increased over time. Finally, at Day 15 (15 days after the final administration of Irinotecan), tumor structure and size were once again the same as in the control group. LGR5, a known cancer stem cell (CSC) marker for CRC, was highly expressed on protruding structures observed from Duct (Flattened) during their transformation into Duct (Columnar) and Cribriform. In addition, these LGR5-expressing protruding structures were either Ki67 negative or positive. These results suggest that the formation of protruding structures on Duct (Flattened) is a pivotal first step in the regrowth of tumors.

Antibody to CD137 Activated by Extracellular Adenosine Triphosphate Is Tumor Selective and Broadly Effective In Vivo without Systemic Immune Activation.

Agonistic antibodies targeting CD137 have been clinically unsuccessful due to systemic toxicity. Because conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigens, we exploited extracellular adenosine triphosphate (exATP), which is a hallmark of the tumor microenvironment and highly elevated in solid tumors, as a broadly tumor-selective switch. We generated a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. STA551 demonstrated potent and broad antitumor efficacy against all mouse and human tumors tested and a wide therapeutic window without systemic immune activation in mice. STA551 was well tolerated even at 150 mg/kg/week in cynomolgus monkeys. These results provide a strong rationale for the clinical testing of STA551 against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy. SIGNIFICANCE: Reported CD137 agonists suffer from either systemic toxicity or limited efficacy against antigen-specific cancers. STA551, an antibody designed to agonize CD137 only in the presence of extracellular ATP, inhibited tumor growth in a broad variety of cancer models without any systemic toxicity or dependence on antigen expression.See related commentary by Keenan and Fong, p. 20.This article is highlighted in the In This Issue feature, p. 1.

Continuous formation of small clusters with LGR5-positive cells contributes to tumor growth in a colorectal cancer xenograft model.

New cancer characteristics can be discovered by focusing on the process of tumor formation. Cancer stem cells (CSCs) are a key subpopulation, as they are theorized to be at the apex of the tumor hierarchy. We can better understand their function in the tumor hierarchy by using sectioned samples to observe the growth of tumors from their origins as CSCs. In this study, we evaluated the growth of moderate differentiated colorectal cancer from LGR5-positive cells, which is a CSC marker of colorectal cancer, using xenograft and three-dimensional culture models spatiotemporally. These cells express LGR5 at high levels and show CSC phenotypes. To detect them, we used a previously generated antibody that specifically targets LGR5, and were therefore able to observe LGR5-positive cells aggregating into small clusters (sCLs) over the course of tumor growth. Because these LGR5-expressing sCLs formed continuously during growth mainly in the invasive front, we concluded that the structure must contribute significantly to the expansion of CSCs and to tumor growth overall. We confirmed the formation of sCLs from gland structures using a three-dimensional culture model. In addition, sCLs exhibited upregulated genes related to stress response and partial/hybrid epithelial-mesenchymal transition (EMT), as well as genes reported to be prognosis factors. Finally, sCLs with high LGR5 expression were identified in clinical samples. Based on these results, we elucidate how sCLs are an important contributors to tumor growth and the expansion of CSCs.

Anti-GPRC5D/CD3 Bispecific T-Cell-Redirecting Antibody for the Treatment of Multiple Myeloma.

Although treatment advances over recent decades have significantly improved survival of patients with multiple myeloma, there is still an unmet medical need for more effective treatments. In this study, we identified G-protein-coupled receptor family C group 5 member D (GPRC5D) expression on the surface of malignant cells involved in multiple myeloma, but except for plasma cells and B cells, not at appreciable levels on normal hematopoietic cells and bone marrow progenitors, including hematopoietic stem cells. In addition, we constructed IgG-based anti-GPRC5D/CD3 bispecific T-cell-redirecting antibodies (GPRC5D TRAB), which suppressed the tumor growth of GPRC5D-positive myeloma cells through the activation of T cells in vitro and in vivo in xenograft models. Collectively, these findings suggest that GPRC5D is an antigen specific to multiple myeloma and a potential target of TRAB therapy.

Antitumor activity of chemoendocrine therapy in premenopausal and postmenopausal models with human breast cancer xenografts.

We examined the efficacy of chemoendocrine therapy using capecitabine as a chemotherapeutic agent in premenopausal and postmenopausal models with estrogen receptor (ER)-positive human breast cancer xenografts. Tamoxifen and letrozole were used as endocrine therapeutic agents for premenopausal and postmenopausal models, respectively. The antitumor activity of capecitabine in combination was significantly superior to either monotherapy treatment in both premenopausal (p<0.01) and postmenopausal (p<0.05) models. No increase in toxicity in terms of body weight loss was observed during treatment in either of the xenograft models. In the premenopausal model, the level of thymidine phosphorylase (TP), a key enzyme generating 5-FU from capecitabine, was upregulated (p<0.05) in tumors by tamoxifen but not by letrozole treatment in the postmenopausal model. The combination of 5'-deoxy-5-fluorouridine (5'-DFUR; an intermediate of capecitabine) with 4-hydroxytamoxifen (4-OHT; an active form of tamoxifen) or letrozole was also evaluated in vitro by using estrogen-responsive element (ERE) reporter gene assays aimed to model premenopausal and postmenopausal breast cancer. Both combinations decreased the number of estrogen-responding cells in a concentration-dependent manner and further analysis by isobolograms revealed a synergistic effect of the combination of 5'-DFUR with 4-OHT, and at least an additive effect of the combination of 5'-DFUR with letrozole. These results suggest that chemoendocrine therapy using capecitabine may be a useful treatment modality for patients with hormone-receptor-positive breast cancer, regardless of the menopausal status and should be explored in clinical trials.

Enhancement of capecitabine efficacy by oxaliplatin in human colorectal and gastric cancer xenografts.

We have evaluated the antitumor activity of XELOX (a combination therapy of capecitabine (Xeloda) and oxaliplatin) in human colorectal and gastric cancer xenograft models. In human colorectal cancer model CXF280, antitumor activity of the combination at two-thirds of the maximum tolerated dose (MTD) was superior to that of each monotherapy at MTD. Furthermore, in human colorectal cancer model COL-05-JCK and human gastric cancer xenograft model GXF 97, the combination also showed at least additive antitumor activity. In addition, toxicity was not augmented with the combination therapy in these three models. As demonstrated using ELISA or immunohistochemistry, oxaliplatin in xenograft model tumors up-regulated the level of thymidine phosphorylase (dThdPase), a key enzyme for the metabolism of capecitabine to 5-fluorouracil. These results suggest that oxaliplatin might potentiate the antitumor activity of capecitabine by up-regulating the tumor level of dThdPase. Based on these results, clinical trials of XELOX against colorectal and gastric cancers are warranted.

Enhancement of antitumor activity of 5'-deoxy-5-fluorouridine (Furtulon) by taxane in human gastric cancer xenografts.

5'-deoxy-5-fluorouridine (5'-DFUR, Furtulon) is activated to 5-fluorouracil (5-FU) by thymidine phosphorylase (dThdPase) highly expressed in many types of tumors. In previous studies, we demonstrated that taxanes (paclitaxel or docetaxel) up-regulated the tumor levels of dThdPase and enhanced the efficacy of 5'-DFUR in human colon and mammary xenograft models. In the present study, combination therapy of 5'-DFUR with taxanes in human gastric cancer xenograft models also showed, at the least, additive anti-tumor activity without significant augmentation of toxicity. Furthermore, paclitaxel up-regulated dThdPase expression in the tumor tissues as confirmed with ELISA and immunohistochemistry. These results suggest taxanes would potentiate the efficacy of 5'-DFUR by up-regulating the tumor levels of dThdPase in gastric xenograft models. Clinical trials of 5'-DFUR in combination with taxane against gastric cancer are warranted.

XELOX (capecitabine plus oxaliplatin): active first-line therapy for patients with metastatic colorectal cancer.

PURPOSE: Capecitabine has demonstrated high efficacy as first-line treatment for metastatic colorectal cancer (MCRC). Oxaliplatin shows synergy with fluorouracil (FU), with little toxicity overlap. The XELOX regimen (capecitabine plus oxaliplatin), established in a previous dose-finding study, should improve on infused oxaliplatin with FU and leucovorin (FOLFOX) regimens. The present studies further characterize efficacy and safety of the XELOX regimen. PATIENTS AND METHODS: The antitumor activity of XELOX was investigated in a colon cancer xenograft model. Patients with MCRC received first-line XELOX in 3-week treatment cycles: intravenous oxaliplatin 130 mg/m(2) (day 1) followed by oral capecitabine 1,000 mg/m(2) twice daily (day 1, evening, to day 15, morning). RESULTS: A preclinical study confirmed that capecitabine has supra-additive activity with oxaliplatin. In the clinical study, 53 of 96 patients (55%) achieved an objective response, and 30 (31%) experienced disease stabilization for >/= 3 months following treatment. After 24 months' minimum follow-up, median time to disease progression (TTP) and median overall survival were 7.7 and 19.5 months, respectively. XELOX safety was predictable and similar to the FOLFOX4 regimen, except that myelosuppression was uncommon with XELOX (grade 3 or 4 neutropenia, 7%). Most adverse events were mild to moderate, the most common being acute sensory neuropathy (85%). Sixty-day, all-cause mortality was 2%. CONCLUSION: XELOX is a highly effective first-line treatment for MCRC. Response rates, TTP, and overall survival are similar to those observed with FU/leucovorin/oxaliplatin combinations. XELOX provides a more convenient regimen, likely to be preferred by both patients and healthcare providers. Capecitabine has the potential to replace FU/LV in combination with oxaliplatin for MCRC.