Digital droplet PCR analysis of organoids generated from mouse mammary tumors demonstrates proof-of-concept capture of tumor heterogeneity.

Breast cancer metastases exhibit many different genetic alterations, including copy number amplifications (CNA). CNA are genetic alterations that are increasingly becoming relevant to breast oncology clinical practice. Here we identify CNA in metastatic breast tumor samples using publicly available datasets and characterize their expression and function using a metastatic mouse model of breast cancer. Our findings demonstrate that our organoid generation can be implemented to study clinically relevant features that reflect the genetic heterogeneity of individual tumors.

Organoid generation from mouse mammary tumors captures the genetic heterogeneity of clinically relevant copy number alterations.

Breast cancer metastases exhibit many different genetic alterations, including copy number amplifications. Using publicly available datasets, we identify copy number amplifications in metastatic breast tumor samples and using our organoid-based metastasis assays, and we validate FGFR1 is amplified in collectively migrating organoids. Because the heterogeneity of breast tumors is increasingly becoming relevant to clinical practice, we demonstrate our organoid method captures genetic heterogeneity of individual tumors.

TK216 targets microtubules in Ewing sarcoma cells.

Ewing sarcoma (EWS) is a pediatric malignancy driven by the EWSR1-FLI1 fusion protein formed by the chromosomal translocation t(11; 22). The small molecule TK216 was developed as a first-in-class direct EWSR1-FLI1 inhibitor and is in phase II clinical trials in combination with vincristine for patients with EWS. However, TK216 exhibits anti-cancer activity against cancer cell lines and xenografts that do not express EWSR1-FLI1, and the mechanism underlying cytotoxicity remains unresolved. We apply a forward-genetics screening platform utilizing engineered hypermutation in EWS cell lines and identify recurrent mutations in TUBA1B, encoding ⍺-tubulin, that prove sufficient to drive resistance to TK216. Using reconstituted microtubule (MT) polymerization in vitro and cell-based chemical probe competition assays, we demonstrate that TK216 acts as an MT destabilizing agent. This work defines the mechanism of cytotoxicity of TK216, explains the synergy observed with vincristine, and calls for a reexamination of ongoing clinical trials with TK216.

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging.

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

TM1385 from Thermotoga maritima functions as a phosphoglucose isomerase via cis-enediol-based mechanism with active site redundancy.

Phosphoglucose isomerases (PGIs) belong to a class of enzymes that catalyze the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. PGIs are crucial in glycolysis and gluconeogenesis pathways and proposed as serving additional extracellular functions in eukaryotic organisms. The phosphoglucose isomerase function of TM1385, a previously uncharacterized protein from Thermotoga maritima, was hypothesized based on structural similarity to established PGI crystal structures and computational docking. Kinetic and colorimetric assays combined with 1H nuclear magnetic resonance (NMR) spectroscopy experimentally confirm that TM1385 is a phosphoglucose isomerase (TmPGI). Evidence of solvent exchange in 1H NMR spectra supports that TmPGI isomerization proceeds through a cis-enediol-based mechanism. To determine which amino acid residues are critical for TmPGI catalysis, putative active site residues were mutated with alanine and screened for activity. Results support that E281 is most important for TmPGI formation of the cis-enediol intermediate, and the presence of either H310 or K422 may be required for catalysis, similar to previous observations from homologous PGIs. However, only TmPGI E281A/Q415A and H310A/K422A double mutations abolished activity, suggesting that there are redundant catalytic residues, and Q415 may participate in sugar phosphate isomerization upon E281 mutation. Combined, we propose that TmPGI E281 participates directly in the cis-enediol intermediate step, and either H310 or K422 may facilitate sugar ring opening and closure.