New hormone receptor-positive breast cancer mouse cell line mimicking the immune microenvironment of anti-PD-1 resistant mammary carcinoma.

BACKGROUND: Progress in breast cancer (BC) research relies on the availability of suitable cell lines that can be implanted in immunocompetent laboratory mice. The best studied mouse strain, C57BL/6, is also the only one for which multiple genetic variants are available to facilitate the exploration of the cancer-immunity dialog. Driven by the fact that no hormone receptor-positive (HR+) C57BL/6-derived mammary carcinoma cell lines are available, we decided to establish such cell lines. METHODS: BC was induced in female C57BL/6 mice using a synthetic progesterone analog (medroxyprogesterone acetate, MPA) combined with a DNA damaging agent (7,12-dimethylbenz[a]anthracene, DMBA). Cell lines were established from these tumors and selected for dual (estrogen+progesterone) receptor positivity, as well as transplantability into C57BL/6 immunocompetent females. RESULTS: One cell line, which we called B6BC, fulfilled these criteria and allowed for the establishment of invasive estrogen receptor-positive (ER+) tumors with features of epithelial to mesenchymal transition that were abundantly infiltrated by myeloid immune populations but scarcely by T lymphocytes, as determined by single-nucleus RNA sequencing and high-dimensional leukocyte profiling. Such tumors failed to respond to programmed cell death-1 (PD-1) blockade, but reduced their growth on treatment with ER antagonists, as well as with anthracycline-based chemotherapy, which was not influenced by T-cell depletion. Moreover, B6BC-derived tumors reduced their growth on CD11b blockade, indicating tumor sustainment by myeloid cells. The immune environment and treatment responses recapitulated by B6BC-derived tumors diverged from those of ER+ TS/A cell-derived tumors in BALB/C mice, and of ER- E0771 cell-derived and MPA/DMBA-induced tumors in C57BL/6 mice. CONCLUSIONS: B6BC is the first transplantable HR+ BC cell line derived from C57BL/6 mice and B6BC-derived tumors recapitulate the complex tumor microenvironment of locally advanced HR+ BC naturally resistant to PD-1 immunotherapy.

Paradoxical implication of BAX/BAK in the persistence of tetraploid cells.

Pro-apoptotic multi-domain proteins of the BCL2 family such as BAX and BAK are well known for their important role in the induction of mitochondrial outer membrane permeabilization (MOMP), which is the rate-limiting step of the intrinsic pathway of apoptosis. Human or mouse cells lacking both BAX and BAK (due to a double knockout, DKO) are notoriously resistant to MOMP and cell death induction. Here we report the surprising finding that BAX/BAK DKO cells proliferate less than control cells expressing both BAX and BAK (or either BAX or BAK) when they are driven into tetraploidy by transient exposure to the microtubule inhibitor nocodazole. Mechanistically, in contrast to their BAX/BAK-sufficient controls, tetraploid DKO cells activate a senescent program, as indicated by the overexpression of several cyclin-dependent kinase inhibitors and the activation of ß-galactosidase. Moreover, DKO cells manifest alterations in ionomycin-mobilizable endoplasmic reticulum (ER) Ca2+ stores and store-operated Ca2+ entry that are affected by tetraploidization. DKO cells manifested reduced expression of endogenous sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (Serca2a) and transfection-enforced reintroduction of Serca2a, or reintroduction of an ER-targeted variant of BAK into DKO cells reestablished the same pattern of Ca2+ fluxes as observed in BAX/BAK-sufficient control cells. Serca2a reexpression and ER-targeted BAK also abolished the tetraploidy-induced senescence of DKO cells, placing ER Ca2+ fluxes downstream of the regulation of senescence by BAX/BAK. In conclusion, it appears that BAX/BAK prevent the induction of a tetraploidization-associated senescence program. Speculatively, this may contribute to the low incidence of cancers in BAX/BAK DKO mice and explain why human cancers rarely lose the expression of both BAX and BAK.

A novel tool for detecting lysosomal membrane permeabilization by high-throughput fluorescence microscopy.

Lysosomes are placed at the center of cellular trafficking and degradative pathways. They also function as a signaling platform for nutrient sensing and metabolic reprogramming. Lysosomes play crucial roles in cellular adaptation in response to stress and are tightly connected to a variety of cell death modalities. Several stimuli can initiate the permeabilization of the lysosome membrane, thus causing cell death when the cellular adaptive system fail to repair or replace damaged lysosomes. The induction of lysosomal membrane permeabilization (LMP) triggers the rapid translocation of Galectin 3/LGALS3 from the cytosol to the lysosomal lumen, making it a valuable marker of LMP. However, Galectin 3 can also be recruited to damaged endo/phagosomal membranes. To make sure that Galectin 3 labels damaged lysosomes, it is therefore important to verify its colocalization with lysosomal markers such as lysosome-associated membrane protein 1 (LAMP1). Here, we describe a simple, fast and robust protocol that allows the detection of LMP of individual lysosomes in U2OS cells expressing mCherry-tagged Galectin 3 and mGFP-tagged LAMP1. This method permits the high-throughput detection and quantification of damaged lysosomes by fluorescence microscopy. It also offers the advantage of studying, in the same experiment, the alterations in size, shape and subcellular localization of intact and damaged lysosomes.

Involvement of neural crest and paraxial mesoderm in oral mucosal development and healing.

Tissue engineering therapies using adult stem cells derived from neural crest have sought accessible tissue sources of these cells because of their potential pluripotency. In this study, the gingiva and oral mucosa and their associated stem cells were investigated. Biopsies of these tissues produce neither scarring nor functional problems and are relatively painless, and fresh tissue can be obtained readily during different chairside dental procedures. However, the embryonic origin of these cells needs to be clarified, as does their evolution from the perinatal period to adulthood. In this study, the embryonic origin of gingival fibroblasts were determined, including gingival stem cells. To do this, transgenic mouse models were used to track neural crest derivatives as well as cells derived from paraxial mesoderm, spanning from embryogenesis to adulthood. These cells were compared with ones derived from abdominal dermis and facial dermis. Our results showed that gingival fibroblasts are derived from neural crest, and that paraxial mesoderm is involved in the vasculogenesis of oral tissues during development. Our in vitro studies revealed that the neuroectodermal origin of gingival fibroblasts (or gingival stem cells) endows them with multipotential properties as well as a specific migratory and contractile phenotype which may participate to the scar-free properties of the oral mucosa. Together, these results illustrate the high regenerative potential of neural crest-derived stem cells of the oral mucosa, including the gingiva, and strongly support their use in cell therapy to regenerate tissues with impaired healing.

Identification, expansion and characterization of cancer cells with stem cell properties from head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) is a major public health concern. Recent data indicate the presence of cancer stem cells (CSC) in many solid tumors, including HNSCC. Here, we assessed the stem cell (SC) characteristics, including cell surface markers, radioresistance, chromosomal instability, and in vivo tumorigenic capacity of CSC isolated from HNSCC patient specimens. We show that spheroid enrichment of CSC from early and short-term HNSCC cell cultures was associated with increased expression of CD44, CD133, SOX2 and BMI1 compared with normal oral epithelial cells. On immunophenotyping, five of 12 SC/CSC markers were homogenously expressed in all tumor cultures, while one of 12 was negative, four of 12 showed variable expression, and two of the 12 were expressed heterogeneously. We showed that irradiated CSCs survived and retained their self-renewal capacity across different ionizing radiation (IR) regimens. Fluorescence in situ hybridization (FISH) analyses of parental and clonally-derived tumor cells revealed different chromosome copy numbers from cell to cell, suggesting the presence of chromosomal instability in HNSCC CSC. Further, our in vitro and in vivo mouse engraftment studies suggest that CD44+/CD66- is a promising, consistent biomarker combination for HNSCC CSC. Overall, our findings add further evidence to the proposed role of HNSCC CSCs in therapeutic resistance.

Jejunal T Cell Inflammation in Human Obesity Correlates with Decreased Enterocyte Insulin Signaling.

In obesity, insulin resistance is linked to inflammation in several tissues. Although the gut is a very large lymphoid tissue, inflammation in the absorptive small intestine, the jejunum, where insulin regulates lipid and sugar absorption is unknown. We analyzed jejunal samples of 185 obese subjects stratified in three metabolic groups: without comorbidity, suffering from obesity-related comorbidity, and diabetic, versus 33 lean controls. Obesity increased both mucosa surface due to lower cell apoptosis and innate and adaptive immune cell populations. The preferential CD8αß T cell location in epithelium over lamina propria appears a hallmark of obesity. Cytokine secretion by T cells from obese, but not lean, subjects blunted insulin signaling in enterocytes relevant to apical GLUT2 mislocation. Statistical links between T cell densities and BMI, NAFLD, or lipid metabolism suggest tissue crosstalk. Obesity triggers T-cell-mediated inflammation and enterocyte insulin resistance in the jejunum with potential broader systemic implications.