Although heterogeneity of FAP+ Cancer-Associated Fibroblasts (CAF) has been described in breast cancer, their plasticity and spatial distribution remain poorly understood. Here, we analyze trajectory inference, deconvolute spatial transcriptomics at single-cell level and perform functional assays to generate a high-resolution integrated map of breast cancer (BC), with a focus on inflammatory and myofibroblastic (iCAF/myCAF) FAP+ CAF clusters. We identify 10 spatially-organized FAP+ CAF-related cellular niches, called EcoCellTypes, which are differentially localized within tumors. Consistent with their spatial organization, cancer cells drive the transition of detoxification-associated iCAF (Detox-iCAF) towards immunosuppressive extracellular matrix (ECM)-producing myCAF (ECM-myCAF) via a DPP4- and YAP-dependent mechanism. In turn, ECM-myCAF polarize TREM2+ macrophages, regulatory NK and T cells to induce immunosuppressive EcoCellTypes, while Detox-iCAF are associated with FOLR2+ macrophages in an immuno-protective EcoCellType. FAP+ CAF subpopulations accumulate differently according to the invasive BC status and predict invasive recurrence of ductal carcinoma in situ (DCIS), which could help in identifying low-risk DCIS patients eligible for therapeutic de-escalation.
© 2024. The Author(s).

HIV-1 (HIV) infects CD4+ T cells, the gradual depletion of which can lead to AIDS in the absence of antiretroviral therapy (ART). Some cells, however, survive HIV infection and persist as part of the latently infected reservoir that causes recurrent viremia after ART cessation. Improved understanding of the mechanisms of HIV-mediated cell death could lead to a way to clear the latent reservoir. Death induced by survival gene elimination (DISE), an RNA interference (RNAi)-based mechanism, kills cells through short RNAs (sRNAs) with toxic 6-mer seeds (positions 2 to 7 of sRNA). These toxic seeds target the 3' untranslated region (UTR) of mRNAs, decreasing the expression of hundreds of genes critical for cell survival. In most cells under normal conditions, highly expressed cell-encoded nontoxic microRNAs (miRNAs) block access of toxic sRNAs to the RNA-induced silencing complex (RISC) that mediates RNAi, promoting cell survival. HIV has been shown to inhibit the biogenesis of host miRNAs in multiple ways. We now report that HIV infection of cells deficient in miRNA expression or function results in enhanced RISC loading of an HIV-encoded miRNA HIV-miR-TAR-3p, which can kill cells by DISE through a noncanonical (positions 3 to 8) 6-mer seed. In addition, cellular RISC-bound sRNAs shift to lower seed viability. This also occurs after latent HIV provirus reactivation in J-Lat cells, suggesting independence of permissiveness of cells to viral infection. More precise targeting of the balance between protective and cytotoxic sRNAs could provide new avenues to explore novel cell death mechanisms that could be used to kill latent HIV. IMPORTANCE Several mechanisms by which initial HIV infection is cytotoxic to infected cells have been reported and involve various forms of cell death. Characterizing the mechanisms underlying the long-term survival of certain T cells that become persistent provirus reservoirs is critical to developing a cure. We recently discovered death induced by survival gene elimination (DISE), an RNAi-based mechanism of cell death whereby toxic short RNAs (sRNAs) containing 6-mer seed sequences (exerting 6-mer seed toxicity) targeting essential survival genes are loaded into RNA-induced silencing complex (RISC) complexes, resulting in inescapable cell death. We now report that HIV infection in cells with low miRNA expression causes a shift of mostly cellular RISC-bound sRNAs to more toxic seeds. This could prime cells to DISE and is further enhanced by the viral microRNA (miRNA) HIV-miR-TAR-3p, which carries a toxic noncanonical 6-mer seed. Our data provide multiple new avenues to explore novel cell death mechanisms that could be used to kill latent HIV.

HIV-1 (HIV) infects CD4 positive T cells, the gradual depletion of which can lead to the onset of Acquired Immunodeficiency Syndrome (AIDS) in the absence of antiretroviral therapy (ART). Several forms of cell death have been shown to be involved in HIV-mediated killing of either directly infected or bystander cells. It is still unknown, however, why some cells survive HIV infection and persist as part of the latently infected reservoir that reliably causes recurrent viremia upon ART cessation. Improved understanding of the mechanisms of HIV-mediated cell death could inform innovations designed to clear the latent reservoir. “Death Induced by Survival gene Elimination” (DISE) is an RNA interference (RNAi)-based mechanism that kills cells through short (s)RNAs with toxic 6mer seeds (pos. 2-7 of sRNA). These toxic seeds target reverse complementary seed matches in the 3’UTR of mRNA transcripts to decrease expression of hundreds of genes that are critical for cell survival. In most cells under normal conditions, highly expressed cell-encoded non-toxic microRNAs (miRNAs) block access of toxic sRNAs to the RNA-induced silencing complex (RISC) that mediates RNAi, promoting cell survival. We now report that infection of cells with HIV results in RISC-loading of an HIV-encoded miRNA, v-miRNA HIV-miR-TAR-3p, which kills cells by DISE through a noncanonical (pos. 3-8) 6mer seed. In addition, cellular RISC bound sRNAs shift to lower seed viability. Both these effects also occur with latent HIV provirus reactivation in J-Lat cells, a well-established cell model of HIV latency. Cells lacking expression of miRNA biogenesis genes Drosha, Dicer and Exportin 5 cannot produce protective miRNAs and therefore do not block RISC loading of the v-miRNA HIV-miR-TAR-3p. These mutant cells, as well as cells lacking expression of the RISC component Ago2, are hypersensitive to cell death via DISE induced by HIV infection. More precise targeting of the balance between protective and cytotoxic sRNAs could specifically and transiently increase silencing of cell survival genes to increase DISE. This could be a new addition to a “shock and kill” strategy to enhance depletion of the provirus reservoir during suppressive ART.

Cancer-associated fibroblasts (CAF) are heterogeneous with multiple functions in breast cancer. Recently, we identified a specific CAF subpopulation (referred to as CAF-S1), which promotes immunosuppression and immunotherapy resistance.
Here, by studying a large collection of human samples, we highlight the key function of CD73/NT5E in CAF-S1-mediated immunosuppression in breast cancer. We first reveal that CD73 protein level specifically accumulates in CAF-S1 in breast cancer patients. Interestingly, infiltration of regulatory T lymphocytes (Tregs) is significantly correlated with CD73 expression in stroma but not in epithelium, indicating that CD73 contributes to immunosuppression when expressed in CAF-S1 and not in tumor cells. By performing functional assays based on relevant systems using primary CAF-S1 isolated from patients, we demonstrate that CAF-S1 increase the content in both PD-1+ and CTLA-4+ Tregs. Importantly, the use of a blocking anti-CD73 antibody on CAF-S1 reduces CAF-S1-mediated immunosuppression by preventing expression of these immune checkpoints on Tregs.
Our data support the potential clinical benefit of using both anti-CD73 and immune-checkpoint inhibitors in breast cancer patients for inhibiting CAF-S1-mediated immunosuppression and enhancing anti-tumor immune response.

The capacity of bone marrow mesenchymal stromal cells (BMSCs) to be induced into chondrocytes has drawn much attention for cell-based cartilage repair. BMSCs represent a small proportion of cells of the bone marrow stromal compartment and, thus, culture expansion is a necessity for therapeutic use. However, there is no consensus on how BMSCs should be isolated nor expanded to maximize their chondrogenic potential. During embryonic development pluripotent stem cells differentiate into chondrocytes and form cartilage in a hypoxic microenvironment.
Freshly harvested human BMSCs were isolated and expanded from the aspirates of six donors, under either hypoxic conditions (3% O2) or normoxic conditions (21% O2). A colony-forming unit fibroblastic (Cfu-f) assay was used to determine the number of cell colonies developed from each donor. BMSCs at passage 2 (P2) were characterized by flow cytometry for the phenotypic expression of cell surface markers on mesenchymal stem cells. BMSCs at P2 were subsequently cultured in vitro as three-dimensional cell pellets in a defined serum-free chondrogenic medium under normoxic and hypoxic conditions. Chondrogenic differentiation of the BMSCs was characterized by biochemical and histological methods and by quantitative gene-expression analysis.
After 14 days of culture, the number of BMSC colonies developed under hypoxia was generally higher (8% to 38% depending on donor) than under normoxia. BMSCs were positive for the cell surface markers CD13, CD29, CD44, CD73, CD90, CD105 and CD151, and negative for CD34. Regardless of the oxygen tension during pellet culture, hypoxia-expanded BMSC pellets underwent a more robust chondrogenesis than normoxia-expanded BMSC pellets after three weeks of culture, as judged by increased glycosaminoglycan synthesis and Safranin O staining, along with increased mRNA expression of aggrecan, collagen II and Sox9. Hypoxic conditions enhanced the mRNA expression of hypoxia inducible factor-2 alpha (HIF-2α) but suppressed the mRNA expression of collagen X in BMSC pellet cultures regardless of the oxygen tension during BMSC isolation and propagation.
Taken together, our data demonstrate that isolation and expansion of BMSCs under hypoxic conditions augments the chondrogenic potential of BMSCs. This suggests that hypoxia-mediated isolation and expansion of BMSCs may improve clinical applications of BMSCs for cartilage repair.