Late Local Recurrence after Neoadjuvant Therapy and Radical Resection for Locally Advanced Rectal Cancer.

Neoadjuvant radiochemotherapy (RCT) and lately total neoadjuvant therapy (TNT) improved local recurrence rates of rectal cancer significantly compared to total mesorectal excision (TME) alone. Yet the occurrence and impact of late local recurrences after many years appears to be a distinct biological problem. We included n = 188 patients with rectal cancer after RCT and radical resection in this study; n = 38 of which had recurrent disease (sites: local (8.0%), liver (6.4%), lung (3.7%)). We found that 68% of all recurrences developed within the first two years. Four patients, however, experience recurrence >8 years after surgery. Here, we report and characterize four cases of late local recurrence (10% of patients with recurrent disease), suggesting that neoadjuvant therapy in principle delays local recurrence.

Protocol for establishing a coculture with fibroblasts and colorectal cancer organoids.

The tumor microenvironment is essential for mediating drug resistance and tumor progression. Here, we present a coculture system, which enables drug testing of colorectal cancer organoids and fibroblasts without additional matrix components such as Matrigel or basement membrane extracts. First, we describe steps to use a readout for high-throughput drug testing using a luminescence-based viability assay. Second, we detail a readout that uses flow cytometry to distinguish toxic effects on either colorectal cancer organoids or fibroblasts.

Mesenchymal Stromal Cell-Based Therapy.

The use of mesenchymal stromal cells (MSCs) for clinical application is intensively investigated for a variety of areas, such as bone repair, haematological and autoimmune diseases, and solid organ transplantation [...].

Establishing a New Platform to Investigate the Efficacy of Oncolytic Virotherapy in a Human Ex Vivo Peritoneal Carcinomatosis Model.

Oncolytic virotherapy constitutes a promising treatment option for many solid cancers, including peritoneal carcinomatosis (PC), which still represents a terminal stage of many types of tumors. To date, the in vitro efficacy of oncolytic viruses is mostly tested in 2D-cultured tumor cell lines due to the lack of realistic 3D in vitro tumor models. We have investigated the feasibility of virotherapy as a treatment option for PC in a human ex vivo peritoneum co-culture model. Human HT-29 cancer cells stably expressing marker genes GFP and firefly luciferase (GFP/luc) were cultured on human peritoneum and infected with two prototypic oncolytic viruses (GLV-0b347 and MeV-DsRed). Both viral constructs were able to infect HT-29 cells in patient-derived peritoneum with high tumor specificity. Over time, both GFP signal and luciferase activity decreased substantially, thereby indicating successful virus-induced oncolysis. Furthermore, immunohistochemistry stainings showed specific virotherapeutic infections of HT-29 cells and effective tumor cell lysis in infected co-cultures. Thus, the PC model established here provides a clinically relevant screening platform to evaluate the therapeutic efficacy of virotherapeutic compounds and also to investigate, in an autologous setting, the immunostimulatory potential of oncolytic viruses for PC in a unique human model system superior to standard 2D in vitro models.

Pharmacologic Targeting of MMP2/9 Decreases Peritoneal Metastasis Formation of Colorectal Cancer in a Human Ex Vivo Peritoneum Culture Model.

BACKGROUND: Matrix metalloproteinases (MMPs) play a crucial role in tumour initiation, progression, and metastasis, including peritoneal carcinosis (PC) formation. MMPs serve as biomarkers for tumour progression in colorectal cancer (CRC), and MMP overexpression is associated with advanced-stage metastasis and poor survival. However, the molecular mechanisms of PC from CRC remain largely unclear. METHODS: We investigated the role of MMPs during peritoneal colonisation by CRC cell lines in a human ex vivo peritoneum model and in patient-derived CRC and corresponding PC samples. MMP2 and MMP9 were inhibited using the small-molecule inhibitors batimastat and the specific MMP2/9 inhibitor III. RESULTS: MMP2 and MMP9 were strongly upregulated in patient-derived samples and following peritoneal colonisation by CRC cells in the ex vivo model. MMP inhibition with batimastat reduced colonisation of HT29 and Colo205 cells by 36% and 68%, respectively (p = 0.0073 and p = 0.0002), while MMP2/9 inhibitor III reduced colonisation by 50% and 41%, respectively (p = 0.0003 and p = 0.0051). Fibronectin cleavage was enhanced in patient-derived samples of PC and during peritoneal colonisation in the ex vivo model, and this was inhibited by MMP2/9 inhibition. CONCLUSION: MMPs were upregulated in patient-derived samples and during peritoneal attachment of CRC cell lines in our ex vivo model. MMP2/9 inhibition prevented fibronectin cleavage and peritoneal colonisation by CRC cells. MMP inhibitors might thus offer a potential treatment strategy for patients with PC.

How to Make Sense out of 75,000 Mesenchymal Stromal Cell Publications?

Mesenchymal stromal cells have been the subject of an expanding number of studies over the past decades. Today, over 75,000 publications are available that shine light on the biological properties and therapeutic effects of these versatile cells in numerous pre-clinical models and early-phase clinical trials. The massive number of papers makes it hard for researchers to comprehend the whole field, and furthermore, they give the impression that mesenchymal stromal cells are wonder cells that are curative for any condition. It is becoming increasingly difficult to dissect how and for what conditions mesenchymal stromal cells exhibit true and reproducible therapeutic effects. This article tries to address the question how to make sense of 75,000, and still counting, publications on mesenchymal stromal cells.

A human ex vivo coculture model to investigate peritoneal metastasis and innovative treatment options.

OBJECTIVES: Peritoneal metastasis (PM) is commonly observed in patients with colorectal cancer (CRC). The outcome of these patients is poor, with an average survival of only six months without therapy, which requires a better understanding of PM biology and new treatment strategies. METHODS: We established and characterized a human ex vivo peritoneal model to investigate the mechanisms of peritoneal seeding and possible treatment options. For this, CRC cell lines and patient-derived tumor organoids were cultured together with human peritoneum to investigate the invasion of malignant cells and the effects of local chemotherapy. RESULTS: Fresh human peritoneum was cultured for up to three weeks in a stainless steel ring system, allowing for survival of all peritoneal structures. Peritoneal cell survival was documented by light microscopy and immunohistochemical staining. Further, immunohistological characterization of the tissue revealed CD3-positive T-lymphocytes and vimentin-positive fibroblasts within the peritoneum. In addition, extracellular matrix components (collagens, matrix metalloproteinases) were localized within the tissue. Coculture with CRC cell lines and patient-derived CRC organoids revealed that cancer cells grew on the peritoneum and migrated into the tissue. Coculture with CRC cells confirmed that hyperthermal treatment at 41 °C for 90 min significantly enhanced the intracellular entry of doxorubicin. Moreover, treatment with mitomycin C under hyperthermic conditions significantly reduced the amount of cancer cells within the peritoneum. CONCLUSIONS: This human ex vivo peritoneal model provides a stringent and clinically relevant platform for the investigation of PM and for further elucidation of possible treatment options.

Three dimensional cultivation increases chemo- and radioresistance of colorectal cancer cell lines.

Although 2D cell cultures are commonly used to predict therapy response, it has become clear that 3D cultures may better mimic the in vivo situation and offer the possibility of tailoring translational clinical approaches. Here, we compared the response of 2D and 3D colorectal cancer (CRC) cell lines to irradiation and chemotherapy. Classic 2D cultures and 3D spheroids of CRC cell lines (CaCo2, Colo205, HCT116, SW480) were thoroughly established, then irradiated with doses of 1, 4, or 10 Gy, using a clinical-grade linear accelerator. The response was assessed by immunohistochemistry, flow cytometry, and TUNEL assays. Upon irradiation, CRC 3D spheroids were morphologically altered. After irradiation with 10 Gy, annexin V/PI staining revealed a 1.8- to 4-fold increase in the apoptosis rate in the 2D cell cultures (95% CI 3.24±0.96), and a 1.5- to 2.4-fold increase in the 3D spheroids (95% CI 1.56±0.41). Irradiation with 1 Gy caused 3- and 4-fold increases in TUNEL positive cells in the CaCo2 and HCT116 (p = 0.01) 2D cultures, respectively, compared with a 2-fold increase in the 3D spheroids. Furthermore, the 2D and 3D cultures responded differently to chemotherapy; the 3D cultures were more resistant to 5-FU and cisplatin, but not to doxorubicin and mitomycin C, than the 2D cultures. Taken together, CRC cells cultured as 3D spheroids displayed markedly higher resistance to irradiation therapy and selected chemotherapeutic drugs than 2D cultures. This in vitro difference must be considered in future approaches for determining the ideal in vitro systems that mimic human disease.

A subgroup of pleural mesothelioma expresses ALK protein and may be targetable by combined rapamycin and crizotinib therapy.

Malignant pleural mesothelioma (MPM) is a neoplasm with inferior prognosis and notorious chemotherapeutic resistance. Targeting aberrantly overexpressed kinases to cure MPM is a promising therapeutic strategy. Here, we examined ALK, MET and mTOR as potential therapeutic targets and determined the combinatorial efficacy of ALK and mTOR targeting on tumor cell growth in vivo. First, ALK overexpression, rearrangement and mutation were studied in primary MPM by qRT-PCR, FISH, immunohistochemistry and sequence analysis; mTOR and MET expression by qRT-PCR and immunohistochemistry. Overexpression of full-length ALK transcripts was observed in 25 (19.5%) of 128 primary MPM, of which ten expressed ALK protein. ALK overexpression was not associated with gene rearrangement, amplification or kinase-domain mutation. mTOR protein was detected in 28.7% MPM, co-expressed with ALK or MET in 5% and 15% MPM, respectively. The ALK/MET inhibitor crizotinib enhanced the anti-tumor effect of the mTOR-inhibitor rapamycin in a patient-derived MPM xenograft with co-activated ALK/mTOR: combined therapy achieved tumor shrinkage in 4/5 tumors and growth stagnation in one tumor. Treatment effects on proliferation, apoptosis, autophagy and pathway signaling were assessed using Ki-67 immunohistochemistry, TUNEL assay, LC3B immunofluorescence, and immunoblotting. Co-treatment significantly suppressed cell proliferation and induced autophagy and caspase-independent, necrotic cell death. Rapamycin/crizotinib simultaneously inhibited mTORC1 (evidenced by S6 kinase and RPS6 dephosphorylation) and ALK signaling (ALK, AKT, STAT3 dephosphorylation), and crizotinib suppressed the adverse AKT activation induced by rapamycin. In conclusion, co-treatment with rapamycin and crizotinib is effective in suppressing MPM tumor growth and should be further explored as a therapeutic alternative in mesothelioma.

A novel role of the organizer gene Goosecoid as an inhibitor of Wnt/PCP-mediated convergent extension in Xenopus and mouse.

Goosecoid (Gsc) expression marks the primary embryonic organizer in vertebrates and beyond. While functions have been assigned during later embryogenesis, the role of Gsc in the organizer has remained enigmatic. Using conditional gain-of-function approaches in Xenopus and mouse to maintain Gsc expression in the organizer and along the axial midline, neural tube closure defects (NTDs) arose and dorsal extension was compromised. Both phenotypes represent convergent extension (CE) defects, arising from impaired Wnt/planar cell polarity (PCP) signaling. Dvl2 recruitment to the cell membrane was inhibited by Gsc in Xenopus animal cap assays and key Wnt/PCP factors (RhoA, Vangl2, Prickle, Wnt11) rescued Gsc-mediated NTDs. Re-evaluation of endogenous Gsc functions in MO-mediated gene knockdown frog and knockout mouse embryos unearthed PCP/CE-related phenotypes as well, including cartilage defects in Xenopus and misalignment of inner ear hair cells in mouse. Our results assign a novel function to Gsc as an inhibitor of Wnt/PCP-mediated CE. We propose that in the organizer Gsc represses CE as well: Gsc-expressing prechordal cells, which leave the organizer first, migrate and do not undergo CE like the Gsc-negative notochordal cells, which subsequently emerge from the organizer. In this model, Gsc provides a switch between cell migration and CE, i.e. cell intercalation.