HDAC inhibitors activate lipid peroxidation and ferroptosis in gastric cancer.

Gastric cancer remains among the deadliest neoplasms worldwide, with limited therapeutic options. Since efficacies of targeted therapies are unsatisfactory, drugs with broader mechanisms of action rather than a single oncogene inhibition are needed. Preclinical studies have identified histone deacetylases (HDAC) as potential therapeutic targets in gastric cancer. However, the mechanism(s) of action of HDAC inhibitors (HDACi) are only partially understood. This is particularly true with regard to ferroptosis as an emerging concept of cell death. In a panel of gastric cancer cell lines with different molecular characteristics, tumor cell inhibitory effects of different HDACi were studied. Lipid peroxidation levels were measured and proteome analysis was performed for the in-depth characterization of molecular alterations upon HDAC inhibition. HDACi effects on important ferroptosis genes were validated on the mRNA and protein level. Upon HDACi treatment, lipid peroxidation was found increased in all cell lines. Class I HDACi (VK1, entinostat) showed the same toxicity profile as the pan-HDACi vorinostat. Proteome analysis revealed significant and concordant alterations in the expression of proteins related to ferroptosis induction. Key enzymes like ACSL4, POR or SLC7A11 showed distinct alterations in their expression patterns, providing an explanation for the increased lipid peroxidation. Results were also confirmed in primary human gastric cancer tissue cultures as a relevant ex vivo model. We identify the induction of ferroptosis as new mechanism of action of class I HDACi in gastric cancer. Notably, these findings were independent of the genetic background of the cell lines, thus introducing HDAC inhibition as a more general therapeutic principle.

Molecular Targets for Gastric Cancer Treatment and Future Perspectives from a Clinical and Translational Point of View.

Gastric cancer is a leading cause of cancer death worldwide. Systemic treatment comprising chemotherapy and targeted therapy is the standard of care in advanced/metastatic gastric cancer. Comprehensive molecular characterization of gastric adenocarcinomas by the TCGA Consortium and ACRG has resulted in the definition of distinct molecular subtypes. These efforts have in parallel built a basis for the development of novel molecularly stratified treatment approaches. Based on this molecular characterization, an increasing number of specific genomic alterations can potentially serve as treatment targets. Consequently, the development of promising compounds is ongoing. In this review, key molecular alterations in gastric and gastroesophageal junction cancers will be addressed. Finally, the current status of the translation of targeted therapy towards clinical applications will be reviewed.

PD-1 inhibition in patient derived tissue cultures of human gastric and gastroesophageal adenocarcinoma.

Emerging immunotherapies quest for better patient stratification in cancer treatment decisions. Moderate response rates of PD-1 inhibition in gastric and esophagogastric junction cancers urge for meaningful human model systems that allow for investigating immune responses ex vivo. Here, the standardized patient-derived tissue culture (PDTC) model was applied to investigate tumor response to the PD-1 inhibitor Nivolumab and the CD3/CD28 t-lymphocyte activator ImmunoCultTM. Resident t-lymphocytes, tumor proliferation and apoptosis, as well as bulk gene expression data were analyzed after 72 h of PD-1 inhibition either as monotherapy or combined with Oxaliplatin or ImmunoCultTM. Individual responses to PD-1 inhibition were found ex vivo and combination with chemotherapy or t-lymphocyte activation led to enhanced antitumoral effects in PDTCs. T-lymphocyte activation as well as the addition of pre-cultured peripheral blood mononuclear cells improved PDTC for studying t-lymphocyte and tumor cell communication. These data support the potential of PDTC to investigate immunotherapy ex vivo in gastric and esophagogastric junction cancer.

Examination of ex-vivo viability of human adipose tissue slice culture.

Obesity is associated with significantly higher mortality rates, and excess adipose tissue is involved in respective pathologies. Here we established a human adipose tissue slice cultures (HATSC) model ex vivo. HATSC match the in vivo cell composition of human adipose tissue with, among others, mature adipocytes, mesenchymal stem cells as well as stroma tissue and immune cells. This is a new method, optimized for live imaging, to study adipose tissue and cell-based mechanisms of obesity in particular. HATSC survival was tested by means of conventional and immunofluorescence histological techniques, functional analyses and live imaging. Surgery-derived tissue was cut with a tissue chopper in 500 µm sections and transferred onto membranes building an air-liquid interface. HATSC were cultured in six-well plates filled with Dulbecco's Modified Eagle's Medium (DMEM), insulin, transferrin, and selenium, both with and without serum. After 0, 1, 7 and 14 days in vitro, slices were fixated and analyzed by morphology and Perilipin A for tissue viability. Immunofluorescent staining against IBA1, CD68 and Ki67 was performed to determine macrophage survival and proliferation. These experiments showed preservation of adipose tissue as well as survival and proliferation of monocytes and stroma tissue for at least 14 days in vitro even in the absence of serum. The physiological capabilities of adipocytes were functionally tested by insulin stimulation and measurement of Phospho-Akt on day 7 and 14 in vitro. Viability was further confirmed by live imaging using Calcein-AM (viable cells) and propidium iodide (apoptosis/necrosis). In conclusion, HATSC have been successfully established by preserving the monovacuolar form of adipocytes and surrounding macrophages and connective tissue. This model allows further analysis of mature human adipose tissue biology ex vivo.

Tumor-associated macrophages and individual chemo-susceptibility are influenced by iron chelation in human slice cultures of gastric cancer.

Purpose: Presence of tumor-associated macrophages (TAM) and high levels of ferritin and lipocalin 2 (Lcn2) in the tumor microenvironment are associated with poor prognosis in many types of cancer. Here we investigate whether iron deprivation influences TAM phenotype and chemotherapy resistance in tumor slice cultures (TSC) of gastric cancer. Results: TAM remained morphologically and functionally stable for four DIV. DFO treatment for 72 h decreased ferritin expression in TAM and in the tumor stroma but did not alter Lcn2 expression. TAM phenotype was altered after 72 h of cisplatin or DFO treatment compared with control conditions. Single DFO treatment and combined treatment with cytotoxic drugs significantly increased tumor cell apoptosis in TSC of gastric cancer. Methods: TSC were manufactured by cutting tissue of gastric cancer resection specimens in 350 µm thick slices and cultivating them under standard conditions on a filter membrane, at an air-liquid interface. After 24 h ex vivo, TSC were treated with irinotecan (100 nM) or cisplatin (10 µM) alone and in combination with deferoxamine (DFO; 10 µM, 100 µM), respectively, for 72 h. After four days in vitro (DIV) the TSC were fixated with paraformaldehyde, paraffin embedded and analyzed by immunohistochemistry for apoptosis (cPARP), proliferation (Ki67), TAM (CD68, CD163), ferritin, and Lcn2 expression. Conclusions: TAM are well preserved and can be studied in TSC of gastric cancer. Iron deprivation significantly increased tumor cell apoptosis.

Randomised phase II trial to investigate catumaxomab (anti-EpCAM × anti-CD3) for treatment of peritoneal carcinomatosis in patients with gastric cancer.

BACKGROUND: Peritoneal carcinomatosis (PC) represents an unfavourable prognostic factor for patients with gastric cancer (GC). Intraperitoneal treatment with the bispecific and trifunctional antibody catumaxomab (EpCAM, CD3), in addition to systemic chemotherapy, could improve elimination of PC. METHODS: This prospective, randomised, phase II study investigated the efficacy of catumaxomab followed by chemotherapy (arm A, 5-fluorouracil, leucovorin, oxaliplatin, docetaxel, FLOT) or FLOT alone (arm B) in patients with GC and PC. Primary endpoint was the rate of macroscopic complete remission (mCR) of PC at the time of second diagnostic laparoscopy/laparotomy prior to optional surgery. RESULTS: Median follow-up was 52 months. Out of 35 patients screened, 15 were allocated to arm A and 16 to arm B. mCR rate was 27% in arm A and 19% in arm B (p = 0.69). Severe side effects associated with catumaxomab were nausea, infection, abdominal pain, and elevated liver enzymes. Median progression-free (6.7 vs. 5.4 months, p = 0.71) and overall survival (13.2 vs. 13.0 months, p = 0.97) were not significantly different in both treatment arms. CONCLUSIONS: Addition of catumaxomab to systemic chemotherapy was feasible and tolerable in advanced GC. Although the primary endpoint could not be demonstrated, results are promising for future investigations integrating intraperitoneal immunotherapy into a multimodal treatment strategy.

Individual Susceptibility Analysis Using Patient-derived Slice Cultures of Colorectal Carcinoma.

BACKGROUND: Nonresponse to chemotherapy in colorectal carcinoma (CRC) is still a clinical problem. For most established treatment regimens, no predictive biomarkers are available. Patient-derived tumor slice culture may be a promising ex vivo technology to assess the drug susceptibility in individual tumors. METHODS: Patient-derived slice cultures of CRC specimens were prepared according to a standardized protocol and treated with different concentrations of 5-fluorouracil (5-FU) and an adapted FOLFOX regimen (5-FU and oxaliplatin) to investigate histologic response. Additionally, a semi-automatized readout using fluorescent stain-specific segmentation algorithms for Image J was established to quantify changes in tumor proliferation. Nonresponse to chemotherapy was defined as persisting tumor cell proliferation. RESULTS: Slices treated with 5-FU showed lower tumor cell fractions and dose-dependent alterations of proliferating tumor cells compared with controls (1 µM, Δ +3%; 10 µM, Δ -9%; 100 µM, Δ -15%). Individual tumor samples were examined and differences in chemotherapy susceptibility could be observed. Untreated slice cultures contained an average tumor cell fraction of 31% ± 7%. For all samples, the histopathologic characteristics exhibited some degree of intratumoral heterogeneity with regard to tumor cell morphology and distribution. The original tumor matched the features found in slices at baseline and after 3 days of cultivation. CONCLUSIONS: Patient-derived slice cultures may help to predict response to clinical treatment in individual patients with CRC. Future studies need to address the problem of tumor heterogeneity and evolution. Prospective correlation of ex vivo results with the clinical course of treated patients is warranted.