Despite high expectations for lung tumoroids, they have not been applied in the clinic due to the difficulty of their long-term culture. Here, however, using AO (airway organoid) media developed by the Clevers laboratory, we succeeded in generating 3 lung tumoroid lines for long-term culture (>13 months) from 41 lung cancer cases (primary or metastatic). Use of nutlin-3a was key to selecting lung tumoroids that harbor mutant p53 in order to eliminate normal lung epithelial organoids. Next-generation sequencing (NGS) analysis indicated that each lung tumoroid carried BRAFG469A, TPM3-ROS1 or EGFRL858R/RB1E737*, respectively. Targeted therapies using small molecule drugs (trametinib/erlotinib for BRAFG469A, crizotinib/entrectinib for TPM3-ROS1 and ABT-263/YM-155 for EGFRL858R/RB1E737*) significantly suppressed the growth of each lung tumoroid line. AO media was superior to 3 different media developed by other laboratories. Our experience indicates that long-term lung tumoroid culture is feasible, allowing us to identify NGS-based therapeutic targets and determine the responsiveness to corresponding small molecule drugs.

We previously evaluated Wilms’ tumor gene 1 (WT1) peptide vaccination in a large number of patients with leukemia or solid tumors and have reported that HLA‐A*24:02 restricted, 9‐mer WT1‐235 peptide (CYTWNQMNL) vaccine induces cellular immune responses and elicits WT1‐235‐specific cytotoxic T lymphocytes (CTLs). However, whether this vaccine induces humoral immune responses to produce WT1 antibody remains unknown. Thus, we measured IgG antibody levels against the WT1‐235 peptide (WT1‐235 IgG antibody) in patients with glioblastoma multiforme (GBM) receiving the WT1 peptide vaccine. The WT1‐235 IgG antibody, which was undetectable before vaccination, became detectable in 30 (50.8%) of a total of 59 patients during 3 months of WT1 peptide vaccination. The dominant WT1‐235 IgG antibody subclass was Th1‐type, IgG1 and IgG3. WT1‐235 IgG antibody production was significantly and positively correlated with both progression‐free survival (PFS) and overall survival (OS). Importantly, the combination of WT1‐235 IgG antibody production and positive delayed type‐hypersensitivity (DTH) to the WT1‐235 peptide was a better prognostic marker for long‐term OS than either parameter alone. These results suggested that WT1‐235 peptide vaccination induces not only WT1‐235‐specific CTLs as previously described but also WT1‐235‐specific humoral immune responses associated with antitumor cellular immune response. Our results indicate that the WT1 IgG antibody against the WT1 peptide may be a useful predictive marker, with better predictive performance in combination with DTH to WT1 peptide, and provide a new insight into the antitumor immune response induction in WT1 peptide vaccine‐treated patients.

Thymic epithelial tumors are rare malignancies, and no optimal therapeutic regimen has been defined for patients with advanced disease. Patients with advanced thymic epithelial tumors, which were resistant or intolerable to prior therapies, were eligible for this study. Patients received 9 mer-WT1-derived peptide emulsified with Montanide ISA51 adjuvant via intradermal administration once a week as a monotherapy. After the 3-month-protocol treatment, the treatment was continued mostly at intervals of 2-4 weeks until disease progression or intolerable adverse events occurred. Of the 15 patients enrolled, 11 had thymic carcinoma (TC) and 4 had invasive thymoma (IT). Median period from diagnosis to the start of treatment was 13.3 and 65.5 months for TC and IT, respectively. No patients achieved a complete or partial response. Of the 8 evaluable TC patients, 6 (75.0%) had stable disease (SD) and 2 had progressive disease (PD). Of the 4 evaluable IT patients, 3 (75.0%) had SD and 1 (25.0%) had PD. Median period of monotherapy treatment was 133 and 683 days in TC and IT patients, respectively. No severe adverse events occurred during the 3-month-protocol treatment. As adverse events in long responders, thymoma-related autoimmune complications, pure red cell aplasia and myasthenia gravis occurred in two IT patients. Cerebellar hemorrhage developed in a TC patient complicated with Von Willebrand disease. Induction of WT1-specific immune responses was observed in the majority of the patients. WT1 peptide vaccine immunotherapy may have antitumor potential against thymic malignancies.

The metastatic progression of malignant tumour requires the proteolytic degradation of extracellular matrix components such as type IV collagen, laminin and proteoglycans. Matrix metalloproteinases (MMPs) play important roles in the extracellular matrix degradation; especially the expression of MMP-2/gelatinase A (72-kDa type IV collagenase) correlates closely with the invasive and metastatic phenotype of tumour cell in vivo (Pyke et al, 1992;Stearns and Wang, 1993;Stetler-Stevenson et al, 1993). MMP-2 is biosynthesized as a proenzyme (proMMP-2) as well as other MMPs, and the proteolytic cleavage of propeptide in the zymogen is essential to express enzymic activity in vitro (Okada et al, 1990;Birkedal-Hansen et al, 1993;Itoh et al, 1995). Concerning the activation of proMMPs in vivo, MMP-3/stromelysin-1 is identified as an endogenous activator for proMMP-1/interstitial procollagenase and proMMP-9/progelatinase B (Ito and Nagase, 1988;Ito et al, 1991;Ogata et al, 1992), but not for proMMP-2 (Okada et al, 1990). Baramova et al (1997) and Mazzieri et al (1997) reported the involvement of plasminogen activator/plasmin system in proMMP-2 activation. Recently, membrane type-MMPs, MT1-MMP (Sato et al, 1994), MT2-MMP (Will and Hinzmann, 1995), MT3-MMP (Takino et al, 1995) and MT4-MMP (Puente et al, 1996) have been discovered and reported to specifically activate proMMP-2 on the cell surface. Furthermore, increased expression of MT1-MMP has been shown to correlate with proMMP-2 activation in human malignant tumours in vivo (Yamamoto et al, 1996;Ueno et al, 1997). Thus, MT1-MMP is considered to be a specific in vivo activator for proMMP-2.The activation of proMMP-2 is induced by concanavalin A and 12--tetradecanoylphorbol 13-acetate in human fibroblasts and human fibrosarcoma HT-1080 cells respectively (Ward et al, 1991;Strongin et al, 1993). Lohi et al (1996) also reported that 12--tetradecanoylphorbol 13-acetate and concanavalin A significantly induce the expression of MT1-MMP in HT-1080 cells and human embryonic lung fibroblasts. On the other hand, Seltzer et al (1994) reported that β 1 integrin receptor, possibly α 2 β 1 augments the conversion of 72-kDa proMMP-2 to 62-kDa active form in human fibroblasts cultured in collagen lattices. In addition, calcium ionophores inhibit the phorbol ester-induced proMMP-2 activation Summary Matrix metalloproteinase 2 (MMP-2)/gelatinase A plays an important role in tumour invasion and metastasis. Since MMP-2 is secreted as an inactive form (proMMP-2) from tumour and neighbouring stroma cells, the activation process is necessary to express the enzymic activity for degradation of extracellular matrix components. We herein reported that the activation of proMMP-2 was induced in human squamous carcinoma cells co-cultured with normal human dermal fibroblasts. When A431 cells were co-cultured with human fibroblasts at various cell ratios, 72-kDa proMMP-2 was converted to a 62-kDa active form through the appearance of a 64-kDa intermediate. The activation of proMMP-2 by co-culture wa...