Vascularized tissue on mesh-assisted platform (VT-MAP): a novel approach for diverse organoid size culture and tailored cancer drug response analysis.

This study presents the vascularized tissue on mesh-assisted platform (VT-MAP), a novel microfluidic in vitro model that uses an open microfluidic principle for cultivating vascularized organoids. Addressing the gap in 3D high-throughput platforms for drug response analysis, the VT-MAP can host tumor clusters of various sizes, allowing for precise, size-dependent drug interaction assessments. Key features include capability for forming versatile co-culture conditions (EC, fibroblasts and colon cancer organoids) that enhance tumor organoid viability and a perfusable vessel network that ensures efficient drug delivery and maintenance of organoid health. The VT-MAP enables the culture and analysis of organoids across a diverse size spectrum, from tens of microns to several millimeters. The VT-MAP addresses the inconsistencies in traditional organoid testing related to organoid size, which significantly impacts drug response and viability. Its ability to handle various organoid sizes leads to results that more accurately reflect patient-derived xenograft (PDX) models and differ markedly from traditional in vitro well plate-based methods. We introduce a novel image analysis algorithm that allows for quantitative analysis of organoid size-dependent drug responses, marking a significant step forward in replicating PDX models. The PDX sample from a positive responder exhibited a significant reduction in cell viability across all organoid sizes when exposed to chemotherapeutic agents (5-FU, oxaliplatin, and irinotecan), as expected for cytotoxic drugs. In sharp contrast, PDX samples of a negative responder showed little to no change in viability in smaller clusters and only a slight reduction in larger clusters. This differential response, accurately replicated in the VT-MAP, underscores its ability to generate data that align with PDX models and in vivo findings. Its capacity to handle various organoid sizes leads to results that more accurately reflect PDX models and differ markedly from traditional in vitro methods. The platform's distinct advantage lies in demonstrating how organoid size can critically influence drug response, revealing insights into cancer biology previously unattainable with conventional techniques.

Establishment of patient-derived organotypic tumor spheroid models for tumor microenvironment modeling.

Patient-derived cancer models that reconstitute the characteristics of the tumor microenvironment may facilitate efforts in precision immune-oncology and the discovery of effective anticancer therapies. Organoids that have recently emerged as robust preclinical models typically contain tumor epithelial cells and lack the native tumor immune microenvironment. A patient-derived organotypic tumor spheroid (PDOTS) is a novel and innovative ex vivo system that retains key features of the native tumor immune microenvironment. Here, we established and characterized a series of colorectal cancer PDOTS models for use as a preclinical platform for testing effective immunotherapy and its combinations with other drugs. Partially dissociated (> 100 µm in diameter) tumor tissues were embedded in Matrigel-containing organoid media and subsequently formed into organoid structures within 3 to 7 days of culture. The success rate of growing PDOTS from fresh tissues was ~86%. Morphological analysis showed that the PDOTSs varied in size and structure. Immunofluorescence and flow cytometry analysis revealed that the PDOTSs retained autologous tumor-infiltrating lymphoid cells and tumor-infiltrating lymphoid cells were continually decreased through serial passages. Notably, PDOTSs from tumors from a high-level microsatellite instability-harboring patient were sensitive to anti-PD-1 or anti-PD-L1 antibodies. Our results demonstrate that the PDOTS model in which the tumor immune microenvironment is preserved may represent an advantageous ex vivo system to develop effective immune therapeutics.

Peroxiredoxin I is important for cancer-cell survival in Ras-induced hepatic tumorigenesis.

Peroxiredoxin I (Prx I), an antioxidant enzyme, has multiple functions in human cancer. However, the role of Prx I in hepatic tumorigenesis has not been characterized. Here we investigated the relevance and underlying mechanism of Prx I in hepatic tumorigenesis. Prx I increased in tumors of hepatocellular carcinoma (HCC) patients that aligned with overexpression of oncogenic H-ras. Prx I also increased in H-rasG12V transfected HCC cells and liver tumors of H-rasG12V transgenic (Tg) mice, indicating that Prx I may be involved in Ras-induced hepatic tumorigenesis. When Prx I was knocked down or deleted in HCC-H-rasG12V cells or H-rasG12V Tg mice, cell colony or tumor formation was significantly reduced that was associated with downregulation of pERK pathway as well as increased intracellular reactive oxygen species (ROS) induced DNA damage and cell death. Overexpressing Prx I markedly increased Ras downstream pERK/FoxM1/Nrf2 signaling pathway and inhibited oxidative damage in HCC cells and H-rasG12V Tg mice. In this study, we found Nrf2 was transcriptionally activated by FoxM1, and Prx I was activated by the H-rasG12V/pERK/FoxM1/Nrf2 pathway and suppressed ROS-induced hepatic cancer-cell death along with formation of a positive feedback loop with Ras/ERK/FoxM1/Nrf2 to promote hepatic tumorigenesis.