Background: Patient-derived colorectal cancer (CRC) organoids (PDOs) solely consisting of malignant cells led to major advances in the understanding of cancer treatments. Yet, a major limitation is the absence of cells from the tumor microenvironment, thereby prohibiting potential investigation of treatment responses on immune and structural cells. Currently there are sparse reports describing the interaction of PDOs, cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) in complex primary co-culture assay systems. Methods: Primary PDOs and patient matched CAF cultures were generated from surgical resections. Co-culture systems of PDOs, CAFs and monocytic myeloid cells were set up to recapitulate features seen in patient tumors. Single-cell transcriptomics and flow cytometry was used to show effects of culture systems on TAM populations in the co-culture assays under chemotherapeutic and oncolytic viral treatment. Results: In contrast to co-cultures of tumor cells and monocytes, CAF/monocyte co-cultures and CAF/monocyte/tumor cell triple cultures resulted in a partial differentiation into macrophages and a phenotypic switch, characterized by the expression of major immunosuppressive markers comparable to TAMs in CRC. Oxaliplatin and 5-fluorouracil, the standard-of-care chemotherapy for CRC, induced polarization of macrophages to a pro-inflammatory phenotype comparable to the immunogenic effects of treatment with an oncolytic virus. Monitoring phagocytosis as a functional proxy to macrophage activation and subsequent onset of an immune response, revealed that chemotherapy-induced cell death, but not virus-mediated cell death, is necessary to induce phagocytosis of CRC cells. Moreover, CAFs enhanced the phagocytic activity in chemotherapy treated CRC triple cultures. Conclusions: Primary CAF-containing triple cultures successfully model TAM-like phenotypes ex vivo and allow the assessment of their functional and phenotypic changes in response to treatments following a precision medicine approach.

Rhabdomyosarcoma (RMS) is a mesenchymal tumor of soft tissue in children that originates from a myogenic differentiation defect. Expression of SNAIL transcription factor is elevated in the alveolar subtype of RMS (ARMS), characterized by a low myogenic differentiation status and high aggressiveness. In RMS patients SNAIL level increases with higher stage. Moreover, SNAIL level negatively correlates with MYF5 expression. The differentiation of human ARMS cells diminishes SNAIL level. SNAIL silencing in ARMS cells inhibits proliferation and induces differentiation in vitro, and thereby completely abolishes the growth of human ARMS xenotransplants in vivo. SNAIL silencing induces myogenic differentiation by upregulation of myogenic factors and muscle-specific microRNAs, such as miR-206. SNAIL binds to the MYF5 promoter suppressing its expression. SNAIL displaces MYOD from E-box sequences (CANNTG) that are associated with genes expressed during differentiation and G/C rich in their central dinucleotides. SNAIL silencing allows the re-expression of MYF5 and canonical MYOD binding, promoting ARMS cell myogenic differentiation. In differentiating ARMS cells SNAIL forms repressive complex with histone deacetylates 1 and 2 (HDAC1/2) and regulates their expression. Accordingly, in human myoblasts SNAIL silencing induces differentiation by upregulation of myogenic factors. Our data clearly point to SNAIL as a key regulator of myogenic differentiation and a new promising target for future ARMS therapies.

Constitutive activation of MET signaling impairs myogenic differentiation of rhabdomyosarcoma and promotes its development and progression

While intrinsic changes in aging hematopoietic stem cells (HSCs) are well characterized, it remains unclear how extrinsic factors affect HSC aging. Here, we demonstrate that cells in the niche—endothelial cells (ECs) and CXCL12‐abundant reticular cells (CARs)—highly express the heme‐degrading enzyme, heme oxygenase 1 (HO‐1), but then decrease its expression with age. HO‐1‐deficient animals (HO‐1 ^−/− ) have altered numbers of ECs and CARs that produce less hematopoietic factors. HSCs co‐cultured in vitro with HO‐1 ^−/− mesenchymal stromal cells expand, but have altered kinetic of growth and differentiation of derived colonies. HSCs from young HO‐1 ^−/− animals have reduced quiescence and regenerative potential. Young HO‐1 ^−/− HSCs exhibit features of premature exhaustion on the transcriptional and functional level. HO‐1 ^+/+ HSCs transplanted into HO‐1 ^−/− recipients exhaust their regenerative potential early and do not reconstitute secondary recipients. In turn, transplantation of HO‐1 ^−/− HSCs to the HO‐1 ^+/+ recipients recovers the regenerative potential of HO‐1 ^−/− HSCs and reverses their transcriptional alterations. Thus, HSC‐extrinsic activity of HO‐1 prevents HSCs from premature exhaustion and may restore the function of aged HSCs.

Rhabdomyosarcoma (RMS) is a predominant soft tissue tumor in children and adolescents. For high-grade RMS with metastatic involvement, the 3-year overall survival rate is only 25 to 30%. Thus, understanding the regulatory mechanisms involved in promoting the metastasis of RMS is important. Here, we demonstrate for the first time that the SNAIL transcription factor regulates the metastatic behavior of RMS both in vitro and in vivo. SNAIL upregulates the protein expression of EZRIN and AKT, known to promote metastatic behavior, by direct interaction with their promoters. Our data suggest that SNAIL promotes RMS cell motility, invasion and chemotaxis towards the prometastatic factors: HGF and SDF-1 by regulating RHO, AKT and GSK3β activity. In addition, miRNA transcriptome analysis revealed that SNAIL-miRNA axis regulates processes associated with actin cytoskeleton reorganization. Our data show a novel role of SNAIL in regulating RMS cell metastasis that may also be important in other mesenchymal tumor types and clearly suggests SNAIL as a promising new target for future RMS therapies.

G-quadruplexes (G4) are stacked nucleic acid structures that are stabilized by heme. In cells, they affect DNA replication and gene transcription. They are unwound by several helicases but the composition of the repair complex and its heme sensitivity are unclear. We found that the accumulation of G-quadruplexes is affected by heme oxygenase-1 (Hmox1) expression, but in a cell-type-specific manner: hematopoietic stem cells (HSCs) from Hmox1^−/− mice have upregulated expressions of G4-unwinding helicases (e.g., Brip1, Pif1) and show weaker staining for G-quadruplexes, whereas Hmox1-deficient murine induced pluripotent stem cells (iPSCs), despite the upregulation of helicases, have more G-quadruplexes, especially after exposure to exogenous heme. Using iPSCs expressing only nuclear or only cytoplasmic forms of Hmox1, we found that nuclear localization promotes G4 removal. We demonstrated that the proximity ligation assay (PLA) can detect cellular co-localization of G-quadruplexes with helicases, as well as with HMOX1, suggesting the potential role of HMOX1 in G4 modifications. However, this colocalization does not mean a direct interaction was detectable using the immunoprecipitation assay. Therefore, we concluded that HMOX1 influences G4 accumulation, but rather as one of the proteins regulating the heme availability, not as a rate-limiting factor. It is noteworthy that cellular G4–protein colocalizations can be quantitatively analyzed using PLA, even in rare cells.